F . Secondary grafting of ( GBM44 - GFP + ) isolated from vehicle or prazosin - treated mice bearing tumors . Mice injected with glioblastoma isolated from prazosin - treated mice developed tumors at a lower frequency ( 50 % versus 100 % ) . Moreover , mice injected with glioblastoma isolated from prazosin - treated mice presented a statistically significant survival benefit . Left Panel : Bioluminescent in vivo of secondary grafts . Middle panel : of bioluminescent signals . * P = 0 . 0004 , n = 8 , two - sided Mann - Whitney U - . Right panel : Kaplan - Meyer survival curves of mice bearing secondary graft from glioblastoma isolated from vehicle - prazosin - treated mice .

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F. Secondary grafting of tumor cells (GBM44-GFP+) isolated from vehicle or prazosin-treated mice bearing primary tumors. Mice injected with glioblastoma cells isolated from prazosin-treated mice developed tumors at a lower frequency (50% versus 100%). Moreover, mice injected with glioblastoma cells isolated from prazosin-treated mice presented a statistically significant survival benefit. Left Panel: Bioluminescent in vivo images of secondary grafts. Middle panel: Quantification of the bioluminescent signals. *P=0.0004, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice bearing secondary graft from glioblastoma cells isolated from vehicle- and prazosin-treated mice.


G . Inhibitory effect of low doses of prazosin ( 0 . 15 mg / Kg ) on glioblastoma growth in vivo . Left panel : Bioluminescent in vivo of tumors in mice treated with vehicle or prazosin ( PRZ ) for 45 days . Tumors were initiated with GBM44 GICs ( compare with panel B ) . Middle panel : of bioluminescent signals . * P = 0 . 0007 , n = 8 , two - sided Mann - Whitney U - . Right panel : Kaplan - Meyer survival curves of mice demonstrating a significant survival benefit of a treatment with low doses of prazosin , log - rank Mantel - Cox . treatment period is shaded in gray .

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G. Inhibitory effect of low doses of prazosin (0.15 mg/Kg) on glioblastoma growth in vivo. Left panel: Bioluminescent in vivo images of tumors in mice treated with vehicle or prazosin (PRZ) for 45 days. Tumors were initiated with GBM44 GICs (compare with panel B). Middle panel: Quantification of the bioluminescent signals. *P=0.0007, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice demonstrating a significant survival benefit of a treatment with low doses of prazosin, log-rank Mantel-Cox test. The treatment period is shaded in gray.


A . Viability of GL261 treated with prazosin in vitro . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - .

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A. Viability analysis of GL261 treated with prazosin in vitro. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


C . Prazosin inhibits in vivo tumorgrowth in an immunocompetent model of glioblastoma . Left panel : Bioluminescent in vivo of tumors in C57 / Bl6mice treated with vehicle or prazosin ( PRZ ) for 14 days . Middle panel : of bioluminescent signals . * P = 0 . 0002 , n = 8 , two - sided Mann - Whitney U - . Right panel : Kaplan - Meyer survival curves of mice treated with prazosin ( PRZ ) or vehicle . log - rank Mantel - Cox . treatment period is shaded in gray .

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C. Prazosin inhibits in vivo tumorgrowth in an immunocompetent model of glioblastoma. Left panel: Bioluminescent in vivo images of tumors in C57/Bl6mice treated with vehicle or prazosin (PRZ) for 14 days.Middle panel: Quantification of the bioluminescent signals. *P=0.0002, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice treated with prazosin (PRZ) or vehicle. log-rank Mantel-Cox test. The treatment period is shaded in gray.


D . Hematoxilin / eosin of C57 / Bl6 mice brain coronal sections sacrificed after 14 days of treatment with prazosin . Scale bar = 2 mm .

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D. Hematoxilin/eosin staining of C57/Bl6 mice brain coronal sections sacrificed after 14 days of treatment with prazosin. Scale bar = 2 mm.


E . imaging of GL261 intra - striatal grafts at 1 2 h after an intra - peritoneal injection of BODIPY FL prazosin , green - derivative of prazosin . Scale bar = 2 mm .

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E. Fluorescence imaging of GL261 intra-striatal grafts at 1 and 2 h after an intra-peritoneal injection of BODIPY FL prazosin, the green-fluorescent derivative of prazosin. Scale bar = 2 mm.


A . Immunoblotting for pro - caspase - 3 ( pro - CASP3 ) active caspase - 3 ( CASP3 ) in GICs treated with prazosin ( PRZ ) or vehicle ( V ) demonstrating that prazosin activates caspase - 3 . ZVAD , a caspase inhibitor , prevents prazosin - induced caspase - 3 activation . kDa : kilodaltons .

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A. Immunoblotting for pro-caspase-3 (pro-CASP3) and active caspase-3 (CASP3) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin activates caspase-3. ZVAD, a caspase inhibitor, prevents prazosin-induced caspase-3 activation. kDa: kilodaltons.


B . Immunoblotting for pro - caspase - 9 ( pro - CASP9 ) active caspase - 9 ( CASP9 ) in GICs treated with prazosin ( PRZ ) or vehicle ( V ) demonstrating that prazosin does not activate caspase - 9 .

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B. Immunoblotting for pro-caspase-9 (pro-CASP9) and active caspase-9 (CASP9) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin does not activate caspase-9.


C . Viability of GICs treated with prazosin for 24h in presence or absence of ZVAD , a caspase inhibitor . ZVAD counteracts prazosin - induced GIC . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - .

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C. Viability analysis of GICs treated with prazosin for 24h in the presence or absence of ZVAD, a caspase inhibitor. ZVAD counteracts prazosin-induced GIC death. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


D . Prazosin induces glioblastoma apoptosis in vivo . GFP + GFP - were analyzed from tumors in vehicle or prazosin - treated mice . Annexin V / DAPI was used to identify apoptotic by FACS . Tumors were initiated by GBM44 GICs implantation .

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D. Prazosin induces glioblastoma cell apoptosis in vivo. GFP+ and GFP- cells were analyzed from tumors in vehicle or prazosin-treated mice. Annexin V / DAPI staining was used to identify apoptotic cells by FACS. Tumors were initiated by GBM44 GICs implantation.


E . Dose - response curve of prazosin on GIC survival ( 24h treatment ) . EC50 was determined with curve fit using nonlinear regression .

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E. Dose-response curve of prazosin on GIC survival (24h treatment). EC50 was determined with curve fit using nonlinear regression.


F . Viability of GICs treated with prazosin for 24h in presence or absence of cirazoline , a subtype agonist of α - ARs . Cirazoline did not alter GIC survival counteracted only poorly prazosin - induced GIC . * P < 0 . 05 , n = 4 , two - sided Mann - Whitney U - .

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F. Viability analysis of GICs treated with prazosin for 24h in the presence or absence of cirazoline, a subtype agonist of the α-ARs. Cirazoline did not alter GIC survival and counteracted only poorly prazosin-induced GIC death. *P<0.05, n=4, two-sided Mann-Whitney U-test.


G . binding assays demonstrating absence of prazosin binding sites in GIC preparations . Positive control shows that prazosin binds to preparations of yeast expressing 1 - AR . PRZ : prazosin . CRZ : cirazoline .

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G. Membrane binding assays demonstrating the absence of prazosin binding sites in GIC membrane preparations. Positive control shows that prazosin binds to membrane preparations of yeast expressing 1-AR. PRZ: prazosin. CRZ: cirazoline.


H . Immunoblotting for phosphorylated ERK1 / 2 ( P - ERK1 / 2 ) total ERK1 / 2 following prazosin treatment for 30 min . Prazosin induces ERK1 / 2 phosphorylation in GICs .

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H. Immunoblotting for phosphorylated ERK1/2 (P-ERK1/2) and total ERK1/2 following prazosin treatment for 30 min. Prazosin induces ERK1/2 phosphorylation in GICs.


I . SRE - luciferase reporter activity of GICs treated with prazosin in absence or presence of U0126 ( 10 μM ) , an inhibitor of ERK - activating kinase MEK . U0126 prevented prazosin - induced SRE - luciferase activation in GICs .

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I. SRE-luciferase reporter activity analysis of GICs treated with prazosin in absence or presence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 prevented prazosin-induced SRE-luciferase activation in GICs.


J . Viability of GICs treated with prazosin for 24 h in presence or absence of U0126 ( 10 μM ) , an inhibitor of ERK - activating kinase MEK . U0126 did not counteract prazosin - induced GIC . Blockade of prazosin - induced ERK1 / 2 phosphorylation by U0126 was confirmed by immunoblotting ( insert ) . PRZ : prazosin .

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J. Viability analysis of GICs treated with prazosin for 24 h in the presence or absence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 did not counteract prazosin-induced GIC death. Blockade of prazosin-induced ERK1/2 phosphorylation by U0126 was confirmed by immunoblotting (insert). PRZ: prazosin.


A . PKCδ expression in two - derived GICs NSCs by immunoblotting . GICs express higher of PKCδ than NSCs . kDa : kilodaltons .

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A. PKCδ expression analysis in two patient-derived GICs and NSCs by immunoblotting. GICs express higher levels of PKCδ than NSCs. kDa: kilodaltons.


B . PKCδ by immunostaining ( green ) . Nuclei were stained with DAPI ( blue ) . Enhanced PKCδ punctate expression in prazosin - treated ( PRZ , 5µM ) GICs , as compared to vehicle - treated GICs . PRZ : prazosin . Scale bar : 10 µm .

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B. PKCδ by immunostaining (green). Nuclei were stained with DAPI (blue). Enhanced PKCδ punctate nuclear expression in prazosin-treated (PRZ, 5µM) GICs, as compared to vehicle-treated GICs. PRZ: prazosin. Scale bar: 10 µm.


C . Total PKCδ ( PKCδ 78kDa ) PKCδ ( PKCδ 41kDa ) expression in GICs by immunoblotting . Prazosin ( PRZ ) induces PKCδ cleavage into a 41kDa active fragment . 2 µM rottlerin ( Rott ) , a PKCδ inhibitor , inhibits prazosin - induced PKCδ cleavage . V : vehicle . kDa : kilodaltons .

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C. Total PKCδ (PKCδ 78kDa) and cleaved PKCδ (PKCδ 41kDa) expression analysis in GICs by immunoblotting. Prazosin (PRZ) induces PKCδ cleavage into a 41kDa active fragment. 2 µM rottlerin (Rott), a PKCδ inhibitor, inhibits prazosin-induced PKCδ cleavage. V: vehicle. kDa: kilodaltons.


D . Pro - caspase - 3 ( Pro - CASP3 ) activated caspase - 3 ( CASP3 ) expression in GICs by immunoblotting . 2 µM rottlerin ( Rott ) inhibits prazosin ( PRZ ) - induced caspase - 3 activation . V : vehicle . kDa : kilodaltons .

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D. Pro-caspase-3 (Pro-CASP3) and activated caspase-3 (CASP3) expression analysis in GICs by immunoblotting. 2 µM rottlerin (Rott) inhibits prazosin (PRZ)-induced caspase-3 activation. V: vehicle. kDa: kilodaltons.


E . Inhibition of PKCδ by rottlerin prevents , in a - dependent manner , prazosin - induced GIC . Viability of GICs treated with prazosin for 72 h in presence or absence of rottlerin . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - .

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E. Inhibition of PKCδ by rottlerin prevents, in a concentration-dependent manner, prazosin-induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of rottlerin. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


F . Inhibition of PKCδ by specific anti - PKCδ RACK peptide δV1 . 1 ( 10 μM ) counteracts prazosin - induced GIC . Viability of GICs treated with prazosin for 72 h in presence or absence of δV1 . 1 . * P < 0 . 005 for Prazosin 1 5 µM P < 0 . 001 for Prazosin 10 µM by two - unpaired Student ' s t - , n = 4 .

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F. Inhibition of PKCδ by the specific anti-PKCδ RACK peptide δV1.1 (10 μM) counteracts prazosin-induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of δV1.1. *P < 0.005 for Prazosin 1 and 5 µM and P<0.001 for Prazosin 10 µM by two-tailed unpaired Student's t-test, n=4.


G . Decreased PKCδ expression using shRNA counteracts prazosin - induced GIC . Viability of GICs transduced with scrambled or PKCδ shRNA treated with prazosin for 72 h . * P < 0 . 005 by two - unpaired Student ' s t - , n = 4 .

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G. Decreased PKCδ expression using shRNA counteracts prazosin-induced GIC death. Viability analysis of GICs transduced with scrambled or PKCδ shRNA and treated with prazosin for 72 h. *P < 0.005 by two-tailed unpaired Student's t-test, n=4.


H . Prazosin inhibits AKT phosphorylation in GICs . LY294002 ( LY , 30 µM ) , an inhibitor of PI3K / AKT pathway , was used as positive control . Terazosin , which does not affect GIC viability , does not alter AKT phosphorylation ( see corresponding viability counting in Fig EV4C ) . Phosphorylated AKT ( P - AKT ) total AKT ( AKT ) expression in GICs by immunoblotting . V : vehicle . kDa : kilodaltons .

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H. Prazosin inhibits AKT phosphorylation in GICs. LY294002 (LY, 30 µM), an inhibitor of the PI3K/AKT pathway, was used as positive control. Terazosin, which does not affect GIC viability, does not alter AKT phosphorylation (see the corresponding cell viability counting in Fig EV4C). Phosphorylated AKT (P-AKT) and total AKT (AKT) expression analysis in GICs by immunoblotting. V: vehicle. kDa: kilodaltons.


I . Prazosin ( 10 µM ) does not inhibit AKT phosphorylation in NSCs . by immunoblotting . V : vehicle . kDa : kilodaltons .

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I. Prazosin (10 µM) does not inhibit AKT phosphorylation in NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.


J . β - catenin expression , a downstream target of AKT , is decreased in prazosin - treated GICs , as opposed to NSCs . by immunoblotting . V : vehicle . kDa : kilodaltons .

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J. β-catenin expression, a downstream target of AKT, is decreased in prazosin-treated GICs, as opposed to NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.


K . L . Inhibition of PKCδ using rottlerin ( Rott , 2 µM ) or δV1 . 1 ( 10 µM ) counteracts prazosin inhibition of AKT phosphorylation . by immunoblotting . V : vehicle . kDa : kilodaltons .

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K. L. Inhibition of PKCδ using rottlerin (Rott, 2 µM) or δV1.1 (10 µM) counteracts prazosin inhibition of AKT phosphorylation. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.


B . Bioluminescent in vivo of tumors in mice grafted with GICs expressing either scrambled or PKCδ shRNA . mice were treated with prazosin ( PRZ ) for 45 days . C . of bioluminescent signals showing that decreased expression of PKC counteracts prazosin inhibition of growth . Fold change in total flux represents : total flux after treatment / total flux prior treatment . * P = 0 . 0007 , n = 8 , two - sided Mann - Whitney U - .

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B. Bioluminescent in vivo images of tumors in mice grafted with GICs expressing either scrambled or PKCδ shRNA. All mice were treated with prazosin (PRZ) for 45 days.C. Quantification of the bioluminescent signals showing that decreased expression of PKC counteracts prazosin inhibition of tumor growth. Fold change in total flux represents the ratio: total flux after treatment/total flux prior treatment. *P=0.0007, n=8, two-sided Mann-Whitney U-test.


of four - week - old soil - grown of Col - 0 , snc1 , two independent transgenic lines of SNIPER1 OE into snc1 background . OE stands for overexpression of SNIPER1 . Scale bar = 1 cm . SNIPER1 gene expression in indicated as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . of H . a . Noco2 sporulation in indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . m . ES4326 on four - week - old leaves of indicated genotypes at 0 3 dpi with inoculum of OD600 = 0 . 001 . Error bars represent mean ± SD ( n = 4 for day 0 ; n = 5 for day 3 ) . Three independent experiments were carried out with similar results .

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Morphology of four-week-old soil-grown plants of Col-0, snc1, and two independent transgenic lines of SNIPER1 OE into snc1 background. OE stands for overexpression of SNIPER1. Scale bar = 1 cm. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. Error bars represent mean ± SD (n=4 for day 0; n=5 for day 3). Three independent experiments were carried out with similar results.


of four - week - old soil - grown of two independent transgenic lines of SNIPER1 OE in Col - 0 background . Scale bar = 1 cm . No difference in growth development was observed . SNIPER1 gene expression in indicated as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . of H . a . Noco2 sporulation in indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . t . DC3000 on four - week - old leaves of indicated genotypes at 0 3 dpi with inoculum of OD600 = 0 . 001 . Error bars represent mean ± SD ( n = 4 for day 0 ; n = 5 for day 3 ) . Three independent experiments were carried out with similar results .

O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Morphology of four-week-old soil-grown plants of two independent transgenic lines of SNIPER1 OE in Col-0 background. Scale bar = 1 cm. No difference in growth and development was observed. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.t. DC3000 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. Error bars represent mean ± SD (n=4 for day 0; n=5 for day 3). Three independent experiments were carried out with similar results.


of four - week - old soil - grown of Col - 0 , chs1 - 2 ( A ) , chs2 - 1 ( B ) two independent transgenic lines of SNIPER1 OE into chs1 - 2 ( A ) chs2 - 1 ( B ) backgrounds . were grown at 16 ° C under long day conditions . Scale bar = 1 cm .

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Morphology of four-week-old soil-grown plants of Col-0, chs1-2 (A), chs2-1 (B) and two independent transgenic lines of SNIPER1 OE into chs1-2 (A) and chs2-1 (B) backgrounds. Plants were grown at 16 °C under long day conditions. Scale bar = 1 cm.


of four - week - old soil - grown of Col - 0 , chs3 - 2D ( C ) , snc2 - 1D eds5 npr1 ( D ) two independent transgenic lines of SNIPER1 OE into chs3 - 2D ( C ) snc2 - 1D eds5 npr1 ( D ) backgrounds . Scale bar = 1 cm .

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Morphology of four-week-old soil-grown plants of Col-0, chs3-2D (C), snc2-1D eds5 npr1 (D) and two independent transgenic lines of SNIPER1 OE into chs3-2D (C) and snc2-1D eds5 npr1 (D) backgrounds. Scale bar = 1 cm.


of four - week - old soil - grown of Col - 0 , mekk1 - 5 ndr1 , two independent transgenic lines of SNIPER1 OE into mekk1 - 5 ndr1 background . Scale bar = 1 cm . mekk1 ndr1 double mutant was used instead of mekk1 - 5 single mutant is to overcome sterility . SNIPER1 gene expression in indicated as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . of H . a . Noco2 sporulation in indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . m . ES4326 on four - week - old leaves of indicated genotypes at 0 3 dpi with inoculum of OD600 = 0 . 001 . mn stands for mekk1 - 5 ndr1 double mutant . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results .

O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

Morphology of four-week-old soil-grown plants of Col-0, mekk1-5 ndr1, and two independent transgenic lines of SNIPER1 OE into mekk1-5 ndr1 background. Scale bar = 1 cm. The mekk1 ndr1 double mutant was used instead of the mekk1-5 single mutant is to overcome the sterility. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. mn stands for mekk1-5 ndr1 double mutant. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.


In vitro ubiquitination using E . coli - expressed SNIPER1 - myc ( E3 ) , AtUBC8 ( E2 ) , AtUBA1 ( E1 ) / or HIS - FLAG - Ubiquitin . molecular mass markers are indicated on left ( kDa ) . Asterisk indicates SNIPER1 - myc . Arrow points to His - FLAG - Ub straight lines on right indicate self - ubiquitinated SNIPER1 .

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In vitro ubiquitination assay using E. coli-expressed SNIPER1-myc (E3), AtUBC8 (E2), AtUBA1 (E1) and/or HIS-FLAG-Ubiquitin. The molecular mass markers are indicated on the left (kDa). Asterisk indicates SNIPER1-myc. Arrow points to His-FLAG-Ub and straight lines on the right indicate self-ubiquitinated SNIPER1.


Immunoblot of SNC1 ( A - B ) , SUMM2 - 3HA ( C ) , RPS4HA ( D ) , RPP4myc ( E ) , RPS2HA ( F ) RPM1myc ( G ) in indicated genotypes . For panels , equal loading is shown by Ponceau S of a non - specific band . numbers below represent normalized between of band Ponceau S band ± SD ( n = 3 ) . Molecular mass marker in kilo Daltons is indicated on left . Three independent experiments were carried out with similar results .

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Immunoblot analysis of SNC1 (A-B), SUMM2-3HA (C), RPS4HA (D), RPP4myc (E), RPS2HA (F) and RPM1myc (G) protein levels in the indicated genotypes. For all panels, equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.


Growth of P . s . t . avrRps4 ( A ) , avrRpt2 ( B ) avrRpm1 ( C ) on four - week - old leaves of indicated genotypes at 3 dpi with inoculum of OD600 = 0 . 0001 . Error bars represent mean ± SD ( n = 5 ) . Three independent experiments were carried out with similar results . Appendix Figure S4 shows growth of P . s . t . avrRps4 , avrRpt2 avrRpm1 at 0 dpi . of H . a . Emwa1 sporulation in indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results .

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Growth of P.s.t. avrRps4 (A), avrRpt2 (B) and avrRpm1 (C) on four-week-old leaves of the indicated genotypes at 3 dpi with bacterial inoculum of OD600 = 0.0001. Error bars represent mean ± SD (n=5). Three independent experiments were carried out with similar results. Appendix Figure S4 shows the growth of P.s.t. avrRps4, avrRpt2 and avrRpm1 at 0 dpi. Quantification of H.a. Emwa1 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.


of four - week - old soil - grown of Col - 0 two independent transgenic lines of 35S - SNIPER1H129Y . Scale bar = 1 cm .

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Morphology of four-week-old soil-grown plants of Col-0 and two independent transgenic lines of 35S-SNIPER1H129Y. Scale bar = 1 cm.


PR1 PR2 gene expression in indicated as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results .

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PR1 and PR2 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results.


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Morphology of four-week-old soil-grown plants of Col-0, sniper1-1, sniper2-1, eds1-2, SNIPER1H129Y, SNIPER1H129Y eds1-2, sniper1-1 sniper2-1 eds1-2 and sniper1-c1 sniper2-1 eds1-2. sniper1-1 is an exonic T-DNA insertional mutant. sniper1-c1 is a SNIPER1 knockout mutant containing 807bp deletion that was generated by CRISPR-Cas9 system. Scale bar = 1 cm.


of H . a . Noco2 sporulation in indicated 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Two independent experiments were carried out with similar results .

O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of H.a. Noco2 sporulation in the indicated plants 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Two independent experiments were carried out with similar results.


Immunoblot of RPS4HA ( E ) , SNC1 ( F ) , RPS2HA ( G ) , RPM1myc ( H ) in SNIPER1H129Y background . Equal loading is shown by Ponceau S of a non - specific band . numbers below represent normalized between of band Ponceau S band ± SD ( n = 3 ) . Molecular mass marker in kilo Daltons is indicated on left . Three independent experiments were carried out with similar results .

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Immunoblot of RPS4HA (E), SNC1 (F), RPS2HA (G), and RPM1myc (H) in SNIPER1H129Y background. Equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.


Immunoprecipitation biotinylation of SUMM2NB - FLAG ( A ) , RPP4NB - FLAG ( B ) , or CHS1NB - FLAG ( C ) by SNIPER1 H129Y - HATurboID in N . benthamiana . Two biological repeats were carried out with similar results .

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Immunoprecipitation and biotinylation of SUMM2NB-FLAG (A), RPP4NB-FLAG (B), or CHS1NB-FLAG (C) by SNIPER1 H129Y-HATurboID in N. benthamiana. Two biological repeats were carried out with similar results.


Interaction of SNIPER1 H129Y - CLuc SUMM2NB - NLuc ( D ) , RPP4NB - NLuc ( E ) or CHS1NB - NLuc ( F ) as tested by split - luciferase complementary in N . benthamiana . Three biological repeats with four technical repeats each were carried out with similar results .

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Interaction of SNIPER1 H129Y-CLuc and SUMM2NB-NLuc (D), RPP4NB-NLuc (E) or CHS1NB-NLuc (F) as tested by split-luciferase complementary assay in N. benthamiana. Three biological repeats with four technical repeats each were carried out with similar results.


Ubiquitination of NB domains from SUMM2 ( A ) , RPP4 ( B ) CHS1 ( C ) . Immunoblot of lysates form E . coil strains expressing AtUBA1 - S , MBP - NB - HA , AtUBC8 - S , HIS - FLAG - UBQ10 SNIPER1 - myc , or strains without one of these components . Two biological repeats were carried out with similar results .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O

Ubiquitination of the NB domains from SUMM2 (A), RPP4 (B) and CHS1 (C). Immunoblot of bacterial lysates form E. coil strains expressing AtUBA1-S, MBP-NB-HA, AtUBC8-S, HIS-FLAG-UBQ10 and SNIPER1-myc, or strains without one of these components. Two biological repeats were carried out with similar results.


( G ) Reduced NMDA / AMPA at SC - CA1 synapses of PtenΔC / ΔC mice ( P17 - 21 ) . NMDAR component was obtained 60 ms after stimulation . ( , n = 11 from 8 mice for WT 8 ( 8 ) for ΔC ; decay , n = 12 from 8 mice for WT 11 ( 8 ) for ΔC , * * P < 0 . 01 , ns , not significant , Mann - Whitney U ) . error bars represent . ( H ) Reduced amplitude but not frequency of NMDA - mEPSCs at PtenΔC / ΔC CA1 pyramidal neurons . ( n = 17 from 4 mice for WT 18 ( 4 ) for ΔC , * * P < 0 . 01 , ns , not significant , Mann - Whitney U , Student ' s t - ) . error bars represent . ( I ) Reduced NMDA / AMPA at synapses in layer II / III pyramidal neurons in prelimbic region of mPFC of PtenΔC / ΔC mice ( P20 - 21 ) . NMDAR component was obtained 60 ms after stimulation . ( n = 9 from 3 mice for WT 10 ( 3 ) for ΔC , * P < 0 . 05 , * * P < 0 . 01 , Student ' s t - , Mann - Whitney U ) . error bars represent . Data information : ( C - F ) Results from last 10 - minute recordings are shown in scatter plots .

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(G) Reduced NMDA/AMPA ratio at SC-CA1 synapses of PtenΔC/ΔC mice (P17-21). The NMDAR component was obtained 60 ms after stimulation. (ratio, n = 11 cells from 8 mice for WT and 8 (8) for ΔC; decay, n = 12 cells from 8 mice for WT and 11 (8) for ΔC, **P < 0.01, ns, not significant, Mann-Whitney U test). The error bars represent SEM. (H) Reduced amplitude but not frequency of NMDA -mEPSCs at PtenΔC/ΔC CA1 pyramidal neurons. (n = 17 cells from 4 mice for WT and 18 (4) for ΔC, **P < 0.01, ns, not significant, Mann-Whitney U test, Student's t-test). The error bars represent SEM. (I) Reduced NMDA/AMPA ratio at synapses in layer II/III pyramidal neurons in the prelimbic region of the mPFC of PtenΔC/ΔC mice (P20-21). The NMDAR component was obtained 60 ms after stimulation. (n = 9 cells from 3 mice for WT and 10 (3) for ΔC, *P < 0.05, **P < 0.01, Student's t-test, Mann-Whitney U test). The error bars represent SEM. Data information: (C-F) Results from the last 10-minute recordings are shown in scatter plots.


A ) A volcano plot showing DEGs ( adjusted p value < 0 . 1 ) derived from - Seq results for PtenΔC / ΔC WT mice ( P21 ) . For calculation of adjusted p value , we have employed R package DESeq2 where P ‐ values obtained by Wald are corrected for multiple testing using Benjamini Hochberg method . ( n = 3 mice for WT ΔC ) . See also Dataset EV1 for full - Seq results .

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A) A volcano plot showing DEGs (adjusted p value < 0.1) derived from RNA-Seq results for PtenΔC/ΔC and WT mice (P21). For the calculation of adjusted p value, we have employed the R package DESeq2 where the P‐values obtained by the Wald test are corrected for multiple testing using the Benjamini and Hochberg method. (n = 3 mice for WT and ΔC). See also Dataset EV1 for the full RNA-Seq results.


C ) Immunoblot validation of increase in SLC6A20 in hippocampal lysates of PtenΔC / ΔC mice ( P21 ) . We denoted antibody as ' SLC6A20 ' here , not ' SLC6A20A ' , because this antibody was generated using a synthetic peptide ( YNEPSNNCQKHAI ) that is commonly present in SLC6A20A SLC6A20B , being a pan - SLC6A20 antibody ( ThermoFisher ) . ( n = 3 mice for WT ∆ C , * P < 0 . 05 , Student ' s t - ) . error bars represent .

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C) Immunoblot validation of the increase in SLC6A20 protein levels in hippocampal lysates of PtenΔC/ΔC mice (P21). We denoted the antibody as 'SLC6A20' here, not 'SLC6A20A', because this antibody was generated using a synthetic peptide sequence (YNEPSNNCQKHAI) that is commonly present in SLC6A20A and SLC6A20B, being a pan-SLC6A20 antibody (ThermoFisher). (n = 3 mice for WT and ∆C, *P < 0.05, Student's t-test). The error bars represent SEM.


( D , E ) Increased phosphorylation of β - catenin at Ser - 675 but not at Ser - 552 in nucleus - enriched P1 fraction but not in whole - brain lysates of PtenΔC / ΔC mice ( 3 months ) . Note that total of β - catenin were not changed in P1 fraction or whole - brain lysates . ( n = 4 mice for WT - WB / P1 ΔC - WB / P1 , * P < 0 . 05 , ns , not significant , Student ' s t - ) . error bars represent .

O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D,E) Increased phosphorylation of β-catenin at Ser-675 but not at Ser-552 in the nucleus-enriched P1 fraction but not in whole-brain lysates of PtenΔC/ΔC mice (3 months). Note that total levels of β-catenin were not changed in the P1 fraction or whole-brain lysates. (n = 4 mice for WT-WB/P1 and ΔC-WB/P1, *P < 0.05, ns, not significant, Student's t-test). The error bars represent SEM.


( B , C ) Decreased extracellular of proline glycine in brains of PtenΔC / ΔC mice ( 2 - 4 month ) , as determined by microdialysis in hippocampus ( see Materials Methods for details ) . ( proline , n = 13 mice for WT 8 for ΔC , * * P < 0 . 01 , Student ' s t - ; glycine , n = 9 for WT 5 for ∆ C , * * P < 0 . 01 , Student ' s t - , Mann - Whitney U ) . error bars represent .

O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-TISSUE O B-GENEPROD O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B,C) Decreased extracellular levels of proline and glycine in the brains of PtenΔC/ΔC mice (2-4 month), as determined by microdialysis in the hippocampus (see Materials and Methods for details). (proline, n = 13 mice for WT and 8 for ΔC, **P < 0.01, Student's t-test; glycine, n = 9 for WT and 5 for ∆C, **P < 0.01, Student's t-test, Mann-Whitney U test). The error bars represent SEM.


( B ) Normalization of NMDAR - dependent HFS - LTP at synapses of PtenΔC / ΔC mice ( 4 - 5 ) by DCS treatment ( 20 μM ) , as shown by fEPSP slopes . ( n = 15 slices from 9 mice for WT - V / vehicle , 11 ( 5 ) for WT - D / DCS , 16 ( 10 ) for ΔC - V , 13 ( 7 ) for ΔC - D , * * P < 0 . 01 , ns , not significant , two - way ANOVA with Tukey ' s ) . grey traces represent baseline fEPSP prior to LTP induction . error bars represent .

O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O

(B) Normalization of NMDAR-dependent HFS-LTP at synapses of PtenΔC/ΔC mice (4-5 weeks) by DCS treatment (20 μM), as shown by fEPSP slopes. (n = 15 slices from 9 mice for WT-V/vehicle, 11 (5) for WT-D/DCS, 16 (10) for ΔC-V, 13 (7) for ΔC-D, **P < 0.01, ns, not significant, two-way ANOVA with Tukey's test). The grey traces represent the baseline fEPSP prior to LTP induction. The error bars represent SEM.


( F ) Normalization of NMDAR - dependent HFS - LTP at synapses of PtenΔC / ΔC mice ( 4 - 5 ) by sarcosine treatment ( 750 µM ) , as shown by fEPSP slopes . Note that data for WT - V ΔC - V are identical to those shown in Fig . 4B because whole experiments were performed together ; we generated independent figures for DCS sarcosine results for clear presentation of data . ( n = 15 slices from 9 mice for WT - V / vehicle , 9 ( 4 ) for WT - Sarc / Sarcosine , 16 ( 10 ) for ΔC - V / Vehicle , 18 ( 4 ) for ΔC - S , * P < 0 . 05 , * * * P < 0 . 001 , ns , not significant , two - way ANOVA with Tukey ' s ) . grey traces represent baseline fEPSP prior to LTP induction . error bars represent .

O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O

(F) Normalization of NMDAR-dependent HFS-LTP at synapses of PtenΔC/ΔC mice (4-5 weeks) by sarcosine treatment (750 µM), as shown by fEPSP slopes. Note that the data for WT-V and ΔC-V are identical to those shown in Fig. 4B because the whole experiments were performed together; we generated independent figures for DCS and sarcosine results for the clear presentation of the data. (n = 15 slices from 9 mice for WT-V/vehicle, 9 (4) for WT-Sarc/Sarcosine, 16 (10) for ΔC-V/Vehicle, 18 (4) for ΔC-S, *P < 0.05, ***P < 0.001, ns, not significant, two-way ANOVA with Tukey's test). The grey traces represent the baseline fEPSP prior to LTP induction. The error bars represent SEM.


( D ) PtenΔC / ΔC mice ( 2 - 4 months ) treated with Slc6a20a - ASO display partially normalized of whole - brain glycine , as shown by lack of difference between saline - treated WT ASO - treated PtenΔC / ΔC mice . Note that glycine observed here are ~ 4 times lower than those measured in Fig . 3A , which could be attributable to different mouse ages ( P21 in Fig . 3A vs . 2 - 4 months in Fig . 5D ) , absence presence of ASO injection , or lot - to - lot variation of ELISA kits . ( n = 8 mice for WT - saline , 5 for WT - ASO , 7 for ΔC - saline , 8 for ΔC - ASO , * * P < 0 . 01 , ns , not significant , two - way ANOVA with Tukey ' s ) . error bars represent .

O O O B-GENEPROD O O B-ORGANISM O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) PtenΔC/ΔC mice (2-4 months) treated with Slc6a20a-ASO display partially normalized levels of whole-brain glycine, as shown by the lack of difference between saline-treated WT and ASO-treated PtenΔC/ΔC mice. Note that the glycine levels observed here are ~4 times lower than those measured in Fig. 3A, which could be attributable to different mouse ages (P21 in Fig. 3A vs. 2-4 months in Fig. 5D), absence and presence of ASO injection, or lot-to-lot variation of the ELISA kits. (n = 8 mice for WT-saline, 5 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, **P < 0.01, ns, not significant, two-way ANOVA with Tukey's test). The error bars represent SEM.


( E ) PtenΔC / ΔC mice ( P20 - 37 ) treated with Slc6a20a - ASO display an NMDA / AMPA at hippocampal SC - CA1 synapses that is comparable to that in ASO - treated WT mice . ( n = 11 neurons from 5 mice for WT - ASO , 10 ( 4 ) for ΔC - ASO , ns , not significant , Mann - Whitney ' s U ) . error bars represent .

O O O B-GENEPROD O O B-ORGANISM O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-TISSUE O O B-TISSUE B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O B-CELL_TYPE O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

(E) PtenΔC/ΔC mice (P20-37) treated with Slc6a20a-ASO display an NMDA/AMPA ratio at hippocampal SC-CA1 synapses that is comparable to that in ASO-treated WT mice. (n = 11 neurons from 5 mice for WT-ASO, 10 (4) for ΔC-ASO, ns, not significant, Mann-Whitney's U test). The error bars represent SEM.


( G ) PtenΔC / ΔC mice ( 2 - 4 months ) treated with Slc6a20a - ASO display normal of climbing in Laboras . n = 6 mice for WT - saline , 7 for WT - ASO , 7 for ΔC - saline , 8 for ΔC - ASO , * P < 0 . 05 , * * P < 0 . 01 , ns , not significant , two - way ANOVA with Bonferroni ' s . error bars represent .

O O O B-GENEPROD O O B-ORGANISM O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) PtenΔC/ΔC mice (2-4 months) treated with Slc6a20a-ASO display normal levels of climbing in the Laboras test. n = 6 mice for WT-saline, 7 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, *P < 0.05, **P <0.01, ns, not significant, two-way ANOVA with Bonferroni's test. The error bars represent SEM.


( D ) Increased extracellular of glycine proline in brain of Slc6a20a + / - Slc6a20a - / - mice , as shown by microdialysis analyses . Note that glycine are increased in both Slc6a20a + / - Slc6a20a - / - mice , whereas proline are increased only in Slc6a20a - / - mice . ( glycine , n = 19 mice for WT , 11 for HT , 8 for KO , * * P < 0 . 01 , Mann - Whitney U ; proline , n = 15 mice for WT , 11 for HT , 14 for KO , * * * P < 0 . 001 , ns , not significant , Mann - Whitney U ) . error bars represent .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-TISSUE O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Increased extracellular levels of glycine and proline in the brain of Slc6a20a+/- and Slc6a20a-/- mice, as shown by microdialysis analyses. Note that glycine levels are increased in both Slc6a20a+/- and Slc6a20a-/- mice, whereas proline levels are increased only in Slc6a20a-/- mice. (glycine, n = 19 mice for WT, 11 for HT, 8 for KO, **P < 0.01, Mann-Whitney U test; proline, n = 15 mice for WT, 11 for HT, 14 for KO, ***P < 0.001, ns, not significant, Mann-Whitney U test). The error bars represent SEM.


( B ) Glycine / proline - induced currents for mouse SLC6A20A are dependent on sodium chloride , as shown by suppression of currents when sodium chloride is replaced with choline chloride or sodium gluconate . Each experimental condition was sequentially applied to HEK293T , as shown in Appendix Fig . S7B . indicated currents are average values from an ensemble of multiple ( ~ 20 ) in a single well . ( glycine , n = 21 for untransfected / blank , 17 for human SLC6A20 - V1 , 25 for human SLC6A20 - V2 , 33 for mSLC6A20A , 28 for mSLC6A20B * * * P < 0 . 001 ( relative to buffer not containing glycine ) , two - way ANOVA with Bonferroni ' s ; proline , n = 17 for untransfected , 23 for human SLC6A20 - V1 , 47 for SLC6A20 - V2 , 31 for mSLC6A20A , 31 for mSLC6A20B , * * * P < 0 . 001 , two - way ANOVA with Bonferroni ' s ) . error bars represent .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-ORGANISM B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Glycine/proline-induced currents for mouse SLC6A20A are dependent on sodium chloride, as shown by the suppression of the currents when sodium chloride is replaced with choline chloride or sodium gluconate. Each experimental condition was sequentially applied to HEK293T cells, as shown in Appendix Fig. S7B. The indicated currents are average values from an ensemble of multiple (~20) cells in a single well. (glycine, n = 21 cells for untransfected/blank, 17 for human SLC6A20-V1, 25 for human SLC6A20-V2, 33 for mSLC6A20A, 28 for mSLC6A20B ***P < 0.001 (relative to buffer not containing glycine), two-way ANOVA with Bonferroni's test; proline, n = 17 cells for untransfected, 23 for human SLC6A20-V1, 47 for SLC6A20-V2, 31 for mSLC6A20A, and 31 for mSLC6A20B, ***P < 0.001, two-way ANOVA with Bonferroni's test). The error bars represent SEM.


( A - C ) Immunoblots for indicated in BAT , extensor digitorum longus ( EDL ) , epididymal adipose tissue ( eWAT ) heart from 10 ‐ to 12 ‐ month ( mo ) ‐ old control ( Con ) knock out ( KO ) mice . Arrows depict LC3 ‐ I II .

O O O O O B-EXP_ASSAY O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O O

(A-C) Immunoblots for indicated proteins in BAT, extensor digitorum longus (EDL), epididymal white adipose tissue (eWAT) and heart from 10‐ to 12‐month (mo)‐old control (Con) and knock out (KO) mice. Arrows depict LC3‐I and II.


( D ) ATG5 ATG7 mRNA in indicated tissues ( n = 4 )

O O O B-GENEPROD I-GENEPROD O O O O O O O O O

(D) ATG5 and ATG7 mRNA levels in indicated tissues (n=4)


( E ) immunoblots for indicated in spleen , liver , lung , kidney , mediobasal hypothalamus ( MBH ) , inguinal adipose tissue ( ingWAT ) perinephric fat ( pnWAT ) from 10 ‐ to 12 ‐ mo ‐ old Con KO mice .

O O O B-EXP_ASSAY O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O B-ORGANISM O

(E) immunoblots for indicated proteins in spleen, liver, lung, kidney, mediobasal hypothalamus (MBH), inguinal white adipose tissue (ingWAT) and perinephric fat (pnWAT) from 10‐ to 12‐mo‐old Con and KO mice.


( F ) weights ( wt ) of chow diet ( RD ) ‐ fed ( n = 6 - 29 ) , ( G ) high ‐ fat diet ( HFD ) ‐ fed Con KO mice at indicated ages ( n = 4 - 17 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O

(F) Body weights (wt) of chow diet (RD)‐fed (n=6-29), and (G) high‐fat diet (HFD)‐fed Con and KO mice at indicated ages (n=4-17).


( H ) Total fat lean mass of 4 - 7 mo RD ‐ fed Con KO mice ( n = 8 - 12 ) .

O O O O B-TISSUE O O O O O O O O O O O O B-ORGANISM O O O O O O O O

(H) Total body fat and lean mass of 4-7 mo RD‐fed Con and KO mice (n=8-12).


( I ) BAT wt ( n = 4 - 7 ) , ( J ) gastrocnemius ( GA ) wt ( n = 5 - 7 ) , ( K ) soleus wt ( n = 4 - 7 ) , ( L ) eWAT wt ( n = 5 - 7 ) ( M ) visceral ( Visc ) subcutaneous ( Subc ) fat distribution in 10 ‐ mo ‐ old Con KO mice ( n = 4 ) . Values are mean ± s . e . , * P0 . 05 , * * P0 . 01 , * * * P0 . 001 .

O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) BAT wt (n=4-7), (J) gastrocnemius (GA) wt (n=5-7), (K) soleus wt (n=4-7), (L) eWAT wt (n=5-7) and (M) visceral (Visc) and subcutaneous (Subc) body fat distribution in 10‐mo‐old Con and KO mice (n=4). Values are mean±s.e., *P0.05, **P0.01, ***P0.001.


( A - B ) mRNA for indicated genes in BAT ( n = 4 )

(A-B) mRNA for indicated genes in BAT (n=4)


( c ) mRNA for indicated genes in epididymal adipose tissue ( eWAT ) ( n = 4 )

O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O

(c) mRNA for the indicated genes in epididymal white adipose tissue (eWAT) (n=4)


( D ) immunoblots for indicated in BAT from 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) knock out ( KO ) mice . Arrows depict isoforms .

O O O B-EXP_ASSAY O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O

(D) immunoblots for indicated proteins in BAT from 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice. Arrows depict protein isoforms.


( E ) immunoblots for indicated in eWAT from 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) knock out ( KO ) mice . Arrows depict isoforms .

(E) immunoblots for indicated proteins in eWAT from 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice. Arrows depict protein isoforms.


( F ) Electron ( × 10 , 000 magnification ) of BAT depicting mitochondria from 4 ‐ mo ‐ old Con KO mice . m , mitochondria ; LD , lipid droplet ; n , nucleus .

O O O B-EXP_ASSAY O O O O O O O O B-TISSUE O B-SUBCELLULAR O O O O O O O O B-ORGANISM O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O

(F) Electron micrographs ( × 10,000 magnification) of BAT depicting mitochondria from 4‐mo‐old Con and KO mice. m, mitochondria; LD, lipid droplet; n, nucleus.


( G ) mRNA ( n = 4 ) in BAT from 10 ‐ mo ‐ old Con KO

O O O O O O O O O O B-TISSUE O O O O O O O O

(G) mRNA levels (n=4) in BAT from 10‐mo‐old Con and KO mice


( H ) hematoxylin eosin ( HE ) ( I ) Sirius Red stains in BAT from 10 ‐ mo ‐ old Con KO mice ( n = 3 - 4 ) . Average adipocyte LD , LD number in BAT are shown . Scale bar , 50 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O O O B-ORGANISM O O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O B-SUBCELLULAR O O B-TISSUE O O O O O O O O O

(H) hematoxylin and eosin (HE) and (I) Sirius Red stains in BAT from 10‐mo‐old Con and KO mice (n=3-4). Average adipocyte and LD size, and LD number in BAT are shown. Scale bar, 50 μm.


( J ) BAT mRNA from 4 ‐ mo ‐ old RD ‐ fed Con KO mice cold ‐ challenged for 75 min ( n = 3 ) . Values are mean ± s . e . * * * P0 . 001 .

O O O B-TISSUE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) BAT mRNA levels from 4‐mo‐old RD‐fed Con and KO mice cold‐challenged for 75 min (n=3). Values are mean±s.e. ***P0.001.


( A ) before ( Basal ) during cold exposure ( n = 7 - 8 ) in 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) knock out ( KO )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Body temperature before (Basal) and during cold exposure (n=7-8) in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice


( B ) Oxygen consumption ( VO2 ) , ( C ) carbon dioxide production ( VCO2 ) in 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) knock out ( KO ) mice ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O

(B) Oxygen consumption (VO2), (C) carbon dioxide production (VCO2) in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice (n=4).


( D ) energy expenditure in 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) knock out ( KO ) mice ( n = 4 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O

(D) energy expenditure in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice (n=4)


( E ) VO2 , ( F ) VCO2 ( G ) energy expenditure in 10 ‐ mo ‐ old high ‐ fat diet ( HFD ) ‐ fed Con KO mice ( n = 3 - 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O

(E) VO2, (F) VCO2 and (G) energy expenditure in 10‐mo‐old high‐fat diet (HFD)‐fed Con and KO mice (n=3-5).


( H ) Ambulation ( AMB ) in 10 ‐ mo ‐ old RD ‐ fed ( n = 4 ) HFD ‐ fed Con KO mice ( n = 3 - 5 ) .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O

(H) Ambulation (AMB) in 10‐mo‐old RD‐fed (n=4) and HFD‐fed Con and KO mice (n=3-5).


( I ) Hematoxylin eosin ( HE ) ‐ stained epididymal adipose tissue ( eWAT ) from 6 ‐ mo ‐ old RD ‐ fed Con KO mice . for is shown ( n = 3 ) . Scale bar , 50 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O

(I) Hematoxylin and eosin (HE)‐stained epididymal white adipose tissue (eWAT) from 6‐mo‐old RD‐fed Con and KO mice. Quantification for cell size is shown (n=3). Scale bar, 50 μm.


( J ) Adβ3 mRNA in eWAT from 10 ‐ mo ‐ old RD ‐ fed Con KO mice ( n = 4 ) , in ( K ) inguinal ( ingWAT ) from 4 ‐ mo ‐ oldcold ‐ challenged RD ‐ fed Con KO mice ( n = 3 ) .

O O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-ORGANISM O O O O O O

(J) Adβ3 mRNA in eWAT from 10‐mo‐old RD‐fed Con and KO mice (n=4), and in (K) inguinal (ingWAT) from 4‐mo‐oldcold‐challenged RD‐fed Con and KO mice (n=3).


( L ) Serum ‐ free fatty acid ( FFA ) glycerol ( Gly ) from 4 ‐ to 6 ‐ mo ‐ old Con KO mice untreated ( basal ) or treated with isoproterenol ( Iso ) i . p . for 20 min ( n = 4 - 6 ) , ( M ) from 4 ‐ mo ‐ old RD ‐ fed cold ‐ challenged ( 75 min ) mice ( n = 3 - 4 ) . Values are mean ± s . e . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 .

O O O B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) Serum‐free fatty acid (FFA) and glycerol (Gly) from 4‐ to 6‐mo‐old Con and KO mice untreated (basal) or treated with isoproterenol (Iso) i.p. for 20 min (n=4-6), and (M) from 4‐mo‐old RD‐fed cold‐challenged (75 min) mice (n=3-4). Values are mean±s.e. *P0.05, **P0.01, ***P0.001.


( A , B ) Tmem26 tbx1 mRNA in epididymal eWAT , ( C , D ) ingWAT from Atg7Flox / Floxmice ( 4 months ( mo ) ) injected in BAT with empty ( Con AdV ) or Cre ‐ expressing ( Cre AdV ) adenoviruses cold ‐ exposed or not ( basal ) for 75 min ( n = 4 - 5 ) .

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(A,B) Tmem26 and tbx1 mRNA levels in epididymal eWAT, and (C,D) ingWAT from Atg7Flox/Floxmice (4 months (mo)) injected in BAT with empty (Con AdV) or Cre‐expressing (Cre AdV) adenoviruses and cold‐exposed or not (basal) for 75 min (n=4-5).


( E , F ) hematoxylin eosin ( HE ) ‐ stained ingWAT section from Atg7Flox / Flox mice ( 4 months ( mo ) ) injected in BAT with empty ( Con AdV ) or Cre ‐ expressing ( Cre AdV ) adenoviruses cold ‐ exposed or not ( basal ) for 75 min ( n = 4 - 5 ) .

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(E,F) hematoxylin and eosin (HE)‐stained ingWAT section from Atg7Flox/Flox mice (4 months (mo)) injected in BAT with empty (Con AdV) or Cre‐expressing (Cre AdV) adenoviruses and cold‐exposed or not (basal) for 75 min (n=4-5).


( G ) mRNA for indicated genes in ingWAT from 4 ‐ mo ‐ old cold ‐ exposed Con KO mice ( n = 4 ) .

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(G) mRNA for indicated genes in ingWAT from 4‐mo‐old cold‐exposed Con and KO mice (n=4).


( H , I ) mRNA in eWAT from 10 ‐ mo ‐ old Con KO mice ( n = 4 ) .

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(H,I) mRNA levels in eWAT from 10‐mo‐old Con and KO mice (n=4).


( J ) β ‐ oxidation in eWAT ingWAT from 4 ‐ mo ‐ old Con KO mice ( n = 4 ) .

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(J) β‐oxidation in eWAT and ingWAT from 4‐mo‐old Con and KO mice (n=4).


( K ) mRNA in soleus from 4 ‐ mo ‐ old cold ‐ exposed Con KO mice ( n = 3 - 6 ) .

O O O O O B-TISSUE O O O O O O O O O O O B-ORGANISM O O O O O O O O

(K) mRNA levels in soleus from 4‐mo‐old cold‐exposed Con and KO mice (n=3-6).


( M ) BAT from 4 ‐ mo ‐ old Con KO mice ( n = 4 ) . Values are mean ± s . e . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 .

O O O B-TISSUE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) BAT from 4‐mo‐old Con and KO mice (n=4). Values are mean±s.e. *P0.05, **P0.01, ***P0.001.


( A ) Blood glucose in 6 ‐ to 10 ‐ month ( mo ) ‐ old mice ( n = 12 - 15 ) , ( B ) serum insulin in 10 ‐ mo ‐ old fed control ( Con ) KO mice ( n = 6 ) .

O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O O

(A) Blood glucose in 6‐ to 10‐month (mo)‐old mice (n=12-15), and (B) serum insulin levels in 10‐mo‐old fed control (Con) and KO mice (n=6).


( C ) Glucose tolerance in 10 ‐ mo ‐ old chow diet ( RD ) ‐ fed ( n = 5 ) , ( D ) in 10 ‐ to 12 ‐ mo ‐ old high ‐ fat diet ( HFD ) ‐ fed Con KO mice ( n = 4 - 9 ) .

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(C) Glucose tolerance tests in 10‐mo‐old chow diet (RD)‐fed (n=5), and (D) in 10‐ to 12‐mo‐old high‐fat diet (HFD)‐fed Con and KO mice (n=4-9).


( E ) Insulin tolerance in 10 ‐ mo ‐ old RD ‐ fed Con KO mice ( n = 5 ) .

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(E) Insulin tolerance test in 10‐mo‐old RD‐fed Con and KO mice (n=5).


( F ) Immunoblots for indicated in skeletal muscle from 10 ‐ mo ‐ old Con KO mice .

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(F) Immunoblots for indicated proteins in skeletal muscle from 10‐mo‐old Con and KO mice.


( G ) Hematoxylin eosin ( HE ) ‐ stained gastrocnemius ( GA ) sections from 6 ‐ mo ‐ old Con KO mice . Myofiber cross ‐ sectional ( μm2 ) is shown ( n = 3 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-TISSUE O O O O O O O O O O O O B-ORGANISM O B-SUBCELLULAR O O O O O O O O O O O O O O

(G) Hematoxylin and eosin (HE)‐stained gastrocnemius (GA) sections from 6‐mo‐old Con and KO mice. Myofiber cross‐sectional area (μm2) is shown (n=3).


( H ) Expression of MuRF ‐ 1 Atrogin ‐ 1 in GA TA from 10 ‐ mo ‐ old Con KO mice ( n = 4 )

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O O O O O O O O B-ORGANISM O O O O O

(H) Expression of MuRF‐1 and Atrogin‐1 in GA and TA from 10‐mo‐old Con and KO mice (n=4)


( I ) myogenic genes in GA from 10 ‐ mo ‐ old Con KO mice ( n = 6 ) .

O O O O O O B-TISSUE O O O O O O O O B-ORGANISM O O O O O O

(I) myogenic genes in GA from 10‐mo‐old Con and KO mice (n=6).


( J ) Immunoblots for indicated in tissues from 4 ‐ mo ‐ old Con KO mice ( n = 4 ) .

O O O B-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM O O O O O O

(J) Immunoblots for indicated proteins in tissues from 4‐mo‐old Con and KO mice (n=4).


( K ) mRNA for IRS1 , ( L ) IRS2 in epididymal adipose tissue ( eWAT ) GA from 10 ‐ mo ‐ old Con KO mice ( n = 6 ) .

O O O O O B-GENEPROD O O O O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O B-ORGANISM O O O O O O

(K) mRNA for IRS1, (L) IRS2 in epididymal white adipose tissue (eWAT) and GA from 10‐mo‐old Con and KO mice (n=6).


( A ) Cellomics ( top ) of GFP - LC3 ‐ HEK in full medium ( FM ) or after 2 h in starvation medium ( ES ) with leupeptin ( EL ) . Scale bars = 40 μm . Optimised automated ( bottom left ) programme detecting hoechst ‐ labelled nuclei ( purple ) , outline ( red ) identifies GFP - LC3 spots ( red spots ) in FM EL . Enlarged GFP - LC3 in FM EL ( bottom right ) . Scale bars = 10 μm .

O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

(A) Cellomics images (top) of GFP-LC3‐HEK cells in full medium (FM) or after 2 h in starvation medium (ES) with leupeptin (EL). Scale bars=40 μm. Optimised automated analysis (bottom left) programme detecting hoechst‐labelled nuclei (purple line), cell outline (red line) and identifies GFP-LC3 spots (red spots) in FM and EL. Enlarged GFP-LC3 images in FM and EL (bottom right). Scale bars=10 μm.


( B ) SCPO , STIPO STAPO after indicated siRNA treatment of GFP - LC3 ‐ HEK incubated as in ( A ) . Error bars represent s . e . m . Significance was determined using a two ‐ paired t ‐ compared with RISCfree ( RF ) in EL : SCPO siULK1 , * * * P = 0 . 0002 ; siATG7 , * * P = 0 . 0078 ; siNRBP2 , * * P = 0 . 0088 . STIPO siULK1 , * * * P0 . 0001 ; siATG7 , * * P = 0 . 0060 ; siNRBP2 , * * P = 0 . 0041 . STAPO siULK1 , * * * P0 . 0001 ; siATG7 , * * P = 0 . 0037 ; siNRBP2 , * * P = 0 . 0028 . experiments were performed : RISCfree ( n = 5 ) , siULK1 ( n = 5 ) , siATG7 ( n = 3 ) , siNRBP2 ( n = 3 ) .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) SCPO, STIPO and STAPO after indicated siRNA treatment of GFP-LC3‐HEK cells incubated as in (A). Error bars represent s.e.m. Significance was determined using a two‐tailed paired t‐test compared with RISCfree (RF) in EL: SCPO siULK1, ***P=0.0002; siATG7, **P=0.0078; siNRBP2, **P=0.0088. STIPO siULK1, ***P0.0001; siATG7, **P=0.0060; siNRBP2, **P=0.0041. STAPO siULK1, ***P0.0001; siATG7, **P=0.0037; siNRBP2, **P=0.0028. The experiments were performed: RISCfree (n=5), siULK1 (n=5), siATG7 (n=3), siNRBP2 (n=3).


( D ) Scatter plot of median SCPO Z ‐ scores from siRNA pools in screen that met minimum object ( OC ) cutoff . Dashed lines represent approximate cutoff for 500 best increasers decreasers . RP of known autophagy genes ( ATG4C , ATG14 , RB1CC1 , ATG9A , ATG7 ) are given in Supplementary Table S2 ; AMBRA1 ( FLJ20294 ) , TBC1D25 ( OATL1 ) SH3GLB1 ( BIF ‐ 1 ) in Supplementary Table S1 ; LAMTOR2 has a RP = 708 . 7 RUBICON has a RP = 1126 .

O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O

(D) Scatter plot of median SCPO Z‐scores from siRNA pools in the primary screen that met the minimum object count (OC) cutoff. Dashed lines represent approximate cutoff for 500 best increasers and decreasers. RP of known autophagy genes (ATG4C, ATG14, RB1CC1, ATG9A, ATG7) are given in Supplementary Table S2; AMBRA1 (FLJ20294), TBC1D25 (OATL1) and SH3GLB1 (BIF‐1) in Supplementary Table S1; LAMTOR2 has a RP=708.7 and RUBICON has a RP=1126.


( A ) SCPO , STIPO STAPO for 51 genes validated by two out of four siRNA duplexes reproducing GFP - LC3 spot phenotype , above a cutoff of greater or less than 20 % of control , passing additional criteria ( Supplementary Table 3 ) . Red boxes indicate an increase > 1 . 2 ; pink boxes an increase between 1 . 1 1 . 2 ; boxes are between 0 . 9 1 . 1 ; blue boxes a decrease between 0 . 9 0 . 8 ; blue boxes a decrease to 0 . 8 . Hits underlined were taken forward as three out of four4 hits , ( § ) not chosen because of restricted tissue expression , ( * ) are three out of four when alternate method of normalisation is used .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) SCPO, STIPO and STAPO for the 51 genes validated by two out of four individual siRNA duplexes reproducing the GFP-LC3 spot phenotype, above a cutoff of greater or less than 20% of control, and passing additional criteria (Supplementary Table 3). Red boxes indicate an increase >1.2; pink boxes an increase between 1.1 and 1.2; white boxes are between 0.9 and 1.1; light blue boxes a decrease between 0.9 and 0.8; dark blue boxes a decrease to 0.8. Hits underlined were taken forward as three out of four4 hits, (§) not chosen because of restricted tissue expression, (*) are three out of four when alternate method of normalisation is used.


( B ) Table summarising results from further validation of 20 genes . mRNA scored as ( + ) or ( − ) indicate efficient or poor knockdown , respectively ( see Supplementary Figures S1 S2 ) . Increasers ( left ) or decreasers ( right ) assayed for LC3 lipidation were scored ( + ) for yes , ( − ) for no , ( + / − ) subtle change ( nd ) not done ( Supplementary Figure S3 ) .

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(B) Table summarising results from further validation of 20 genes. mRNA levels scored as (+) or (−) indicate efficient or poor knockdown, respectively (see Supplementary Figures S1 and S2). Increasers (left) or decreasers (right) assayed for LC3 lipidation were scored (+) for yes, (−) for no, (+/−) subtle change and (nd) not done (Supplementary Figure S3).


( C - E ) STIPO results after incubation under different autophagy ‐ inducing conditions following ( C ) SCOC knockdown ( D ) SUPT5H knockdown ( E ) WAC knockdown . siRNA ‐ treated were incubated for indicated time in FL : full medium plus leupeptin ; ES ; EL : ES plus leupeptin ; Torin : 250 nM Torin1 in FL ; Resv : 128 μM Resveratrol in FL ; LiCl : 10 mM LiCl in FM .

O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O

(C-E) STIPO results after incubation under different autophagy‐inducing conditions following (C) SCOC knockdown (D) SUPT5H knockdown and (E) WAC knockdown. siRNA‐treated cells were incubated for the indicated time in FL: full medium plus leupeptin; ES; EL: ES plus leupeptin; Torin: 250 nM Torin1 in FL; Resv: 128 μM Resveratrol in FL; LiCl: 10 mM LiCl in FM.


( B ) Endogenous SCOC shows colocalisation with Atg9 ( red ) TGN46 ( blue ) . HEK293 were fixed permeabilised in saponin . Colocalisation indicated with arrows .

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(B) Endogenous SCOC shows colocalisation with Atg9 (red) and TGN46 (blue). HEK293 cells were fixed and permeabilised in saponin. Colocalisation indicated with arrows.


( C ) Endogenous SCOC ( green top , red bottom ) partially colocalises with endogenous LC3 ( red , top ) GFP - LC3 ( green , bottom ) in HEK293 GFP - LC3 ‐ HEK , respectively , after 2 h ‐ acid starvation . Boxed region is at higher magnification in merge panel on right . Scale bars = 10 μm .

O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_LINE B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

(C) Endogenous SCOC (green top, red bottom) partially colocalises with endogenous LC3 (red, top) and GFP-LC3 (green, bottom) in HEK293 and GFP-LC3‐HEK cells, respectively, after 2 h amino‐acid starvation. Boxed region is at higher magnification in merge panel on right. Scale bars=10 μm.


( A ) SCOC is required for autophagy in HEK293 . Anti ‐ ULK1 , ‐ Actin ‐ LC3 blot after siRNA treatment in HEK293 in FM , ES or EL . Representative blot ; of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) . Significance was determined using a two ‐ paired t ‐ : RF EL versus siSCOC ‐ 03 EL , * P = 0 . 0419 .

O O O B-GENEPROD O O O O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) SCOC is required for autophagy in HEK293 cells. Anti‐ULK1, ‐Actin and ‐LC3 blot after siRNA treatment in HEK293 cells in FM, ES or EL. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RF EL versus siSCOC‐03 EL, *P=0.0419.


( B ) SCOC is required for p62 degradation . p62 were determined by western blot ( Supplementary Figure S5 ) after incubation in FM , ES for 2 or 4 h , or ES with BafilomycinA1 ( ES + BAF ) for 2 4 h . of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) ; RISCfree FM versus RISCfree ES 2 4 h , * * * P > 0 . 0001 * * 0 . 0014 , respectively .

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(B) SCOC is required for p62 degradation. p62 levels were determined by western blot (Supplementary Figure S5) after incubation in FM, ES for 2 or 4 h, or ES with BafilomycinA1 (ES+BAF) for 2 and 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree ES 2 and 4 h, ***P>0.0001 and **0.0014, respectively.


( C ) SCOC - GFP SCOC - myc are reduced by SCOC siRNA duplex ‐ 03 . Anti ‐ GFP , ‐ Myc ‐ Actin blots of HEK293 after treatment with ULK1 , RISCfree or SCOC siRNA overexpression of GFP , SCOC - myc or SCOC - GFP .

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(C) SCOC-GFP and SCOC-myc levels are reduced by SCOC siRNA duplex‐03. Anti‐GFP, ‐Myc and ‐Actin blots of HEK293 cells after treatment with ULK1, RISCfree or SCOC siRNA and overexpression of GFP, SCOC-myc or SCOC-GFP.


( D ) Overexpression of SCOC - myc increases autophagy . Anti ‐ Actin , ‐ LC3 ‐ Myc blots of HEK293 after transfection of pcDNA or SCOC - myc . Representative blot ; of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 5 ) . SCOC - myc EL versus pcDNA EL , * P = 0 . 0215 .

O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O

(D) Overexpression of SCOC-myc increases autophagy. Anti‐Actin, ‐LC3 and ‐Myc blots of HEK293 cells after transfection of pcDNA or SCOC-myc. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=5). SCOC-myc EL versus pcDNA EL, *P=0.0215.


( E ) Endogenous SCOC colocalises with endogenous FEZ1 in HEK293 . Arrows show partial colocalisation . Boxed region is at higher magnification in merge panel on bottom right . Scale bars = 20 μm .

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(E) Endogenous SCOC colocalises with endogenous FEZ1 in HEK293 cells. Arrows show partial colocalisation. Boxed region is at higher magnification in merge panel on bottom right. Scale bars=20 μm.


( A ) Knockdown of FEZ1 enhances LC3 lipidation but does not affect p62 degradation . Anti ‐ Actin ‐ LC3 blot after siRNA treatment in HEK293 . of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) . Significance was determined using a two ‐ paired t ‐ : RISCfree ES versus siFEZ1 ‐ 01 ES , P = 0 . 034 . of p62 / actin ( Supplementary Figure S5 ) performed as in Figure 4B ; error bars represent s . e . m . ( n = 3 ) ; RISCfree FM versus RISCfree 2 4 hP > 0 . 0001 P = 0 . 0014 , respectively . qRT - PCR showing FEZ1 mRNA after indicated knockdown in GFP - LC3 ‐ HEK ( error bars represent s . d . ) .

O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O

(A) Knockdown of FEZ1 enhances LC3 lipidation but does not affect p62 degradation. Anti‐Actin and ‐LC3 blot after siRNA treatment in HEK293 cells. Quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RISCfree ES versus siFEZ1‐01 ES, P=0.034. Quantification of p62/actin ( Supplementary Figure S5) performed as in Figure 4B; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree 2 and 4 hP>0.0001 and P=0.0014, respectively. qRT-PCR showing FEZ1 mRNA levels after indicated knockdown in GFP-LC3‐HEK cells (error bars represent s.d.).


( B ) Overexpression of FEZ1 inhibits LC3 lipidation p62 degradation . Anti ‐ Myc , ‐ Actin ‐ LC3 blot after transfection with pcDNA or Myc - FEZ1 in HEK293 . of LC3II / actin ; error bars represent s . e . m . ( n = 3 ) ; pcDNA ES versus Myc - FEZ1 ES , P = 0 . 0318 . Anti ‐ GFP , ‐ Actin ‐ p62 blot after transfection with GFP or FEZ1 - GFP treatment with ES or ES plus Bafilomycin ( EB ) for 4 h . of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) ; GFP FM versus GFP ES , P = 0 . 0077 ; GFP FM versus GFP EB , P = 0 . 0212 ; GFP ES versus FEZ1 - GFP ES , P = 0 . 048 .

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(B) Overexpression of FEZ1 inhibits LC3 lipidation and p62 degradation. Anti‐Myc, ‐Actin and ‐LC3 blot after transfection with pcDNA or Myc-FEZ1 in HEK293 cells. Quantification of LC3II/actin; error bars represent s.e.m. (n=3); pcDNA ES versus Myc-FEZ1 ES, P=0.0318. Anti‐GFP, ‐Actin and ‐p62 blot after transfection with GFP or FEZ1-GFP and treatment with ES or ES plus Bafilomycin (EB) for 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); GFP FM versus GFP ES, P=0.0077; GFP FM versus GFP EB, P=0.0212; GFP ES versus FEZ1-GFP ES, P=0.048.


( C ) SCOC FEZ1 interact . Left panel shows anti ‐ SCOC ‐ FEZ1 blots after co ‐ expression in HEK293 co ‐ immunoprecipitation of SCOC - myc FEZ1 - GFP with anti ‐ GFP antibody . Input lysate ( Inp ) , unbound supernatant ( Unb ) . Right panel shows anti ‐ FEZ1 ‐ FLAG blots after co ‐ expression in HEK293 co ‐ immunoprecipitation of FLAG - SCOC Myc - FEZ1 with anti ‐ FLAG antibody .

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(C) SCOC and FEZ1 interact. Left panel shows anti‐SCOC and ‐FEZ1 blots after co‐expression in HEK293 cells and co‐immunoprecipitation of SCOC-myc and FEZ1-GFP with anti‐GFP antibody. Input lysate (Inp), unbound supernatant (Unb). Right panel shows anti‐FEZ1 and ‐FLAG blots after co‐expression in HEK293 cells and co‐immunoprecipitation of FLAG-SCOC and Myc-FEZ1 with anti‐FLAG antibody.


( D ) FEZ1 interacts with SCOC in vitro . GST ‐ pulldown assays using in vitro translated 35S ‐ labelled Myc - FEZ1 ( right ) or 35S ‐ labelled Myc ‐ control ( left ) GST or GST - SCOC ( asterisk in bottom panel ) . Bound were detected by autoradiography following SDS - PAGE ( top ) , loading by coomassie blue ( bottom ) .

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(D) FEZ1 interacts with SCOC in vitro. GST‐pulldown assays using in vitro translated 35S‐labelled Myc-FEZ1 (right) or 35S‐labelled Myc‐control (left) and GST or GST-SCOC (asterisk in bottom panel). Bound proteins were detected by autoradiography following SDS-PAGE (top), protein loading by coomassie blue (bottom).


( E ) FEZ1 - SCOC interaction requires evolutionary conserved residues L254 L260 in coiled ‐ coil domain of FEZ1 as demonstrated in GST ‐ pulldown assays with in vitro translated 35S ‐ labelled WT or mutant Myc ‐ FEZ1 GST or GST ‐ SCOC . Autoradiographs of 10 % input bound ( top panels ) Coomassie blue ‐ stained gels of bead ‐ bound GST GST ‐ SCOC used ( bottom panels ) .

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(E) The FEZ1-SCOC interaction requires the evolutionary conserved residues L254 and L260 in the coiled‐coil domain of FEZ1 as demonstrated in GST‐pulldown assays with in vitro translated 35S‐labelled WT or mutant Myc‐FEZ1 and GST or GST‐SCOC. Autoradiographs of 10% input and bound proteins (top panels) and Coomassie blue‐stained gels of bead‐bound GST and GST‐SCOC proteins used (bottom panels).


( A ) FEZ1 interacts with both kinase domain ( 1 - 278 ) proline - serine rich spacer of ULK1 ( 279 - 828 ) . GFP ‐ or Myc ‐ tagged ULK1 fragments were in vitro translated with 35S ‐ methionine subjected to GST ‐ pulldown assays using GST GST - FEZ1 purified from E . coli ( bottom ) . Bound were detected by autoradiography following SDS - PAGE ( top ) .

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(A) FEZ1 interacts with both the kinase domain (1-278) and the proline-serine rich spacer of ULK1 (279-828). GFP‐ or Myc‐tagged ULK1 fragments were in vitro translated with 35S‐methionine and subjected to GST‐pulldown assays using GST and GST-FEZ1 purified from E. coli (bottom). Bound proteins were detected by autoradiography following SDS-PAGE (top).


( B ) ULK1 interacts with FEZ1 . ULK1 was immunoprecipitated from HEK293 lysates with anti ‐ ULK1 bound ULK1 FEZ1 were detected with anti ‐ ULK1 anti ‐ FEZ1 antibodies . Input lysate ( Inp ) .

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(B) ULK1 interacts with FEZ1. ULK1 was immunoprecipitated from HEK293 lysates with anti‐ULK1 and bound ULK1 and FEZ1 were detected with anti‐ULK1 and anti‐FEZ1 antibodies. Input lysate (Inp).


( C ) FEZ1 - GFP Myc - ULK1 interaction is unaffected by ‐ acid starvation . Anti ‐ Myc ‐ GFP blots after HEK293 were co ‐ transfected with FEZ1 - GFP Myc - ULK1 , incubated in FM or ES for 2 h , harvested was immunoprecipitated using anti ‐ GFP antibody .

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(C) The FEZ1-GFP and Myc-ULK1 interaction is unaffected by amino‐acid starvation. Anti‐Myc and ‐GFP blots after HEK293 cells were co‐transfected with FEZ1-GFP and Myc-ULK1, incubated in FM or ES for 2 h, harvested and the complex was immunoprecipitated using anti‐GFP antibody.


( D ) BN - PAGE reveals SCOC FEZ1 are in a . FEZ1 - GFP , FLAG - SCOC Myc - ULK1 wild type or kinase ‐ inactive ULK1K46I ( ULK1KI ) were co ‐ expressed in HEK293 . Anti ‐ GFP , ‐ FLAG ‐ Myc western blots are shown for BN - PAGE gel ( top ) or SDS - PAGE gel ( bottom ) . In GFP FLAG western blots , two different exposures of same BN - PAGE blot are shown spliced together .

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(D) BN-PAGE reveals SCOC and FEZ1 are in a complex. FEZ1-GFP, FLAG-SCOC and Myc-ULK1 wild type or kinase‐inactive ULK1K46I (ULK1KI) were co‐expressed in HEK293 cells. Anti‐GFP, ‐FLAG and ‐Myc western blots are shown for the BN-PAGE gel (top) or the SDS-PAGE gel (bottom). In GFP and FLAG western blots, two different exposures of the same BN-PAGE blot are shown spliced together.


( E ) siRNA depletion of ULK1 does not affect interaction of FEZ1 with SCOC . RF or ULK1 siRNA ‐ treated were co ‐ transfected with FEZ1 - GFP FLAG - SCOC , then incubated immunoprecipitated as in ( C ) . Western blot of lysates for ULK1 , GFP FLAG , immunoprecipitates with GFP FLAG antibodies .

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(E) siRNA depletion of ULK1 does not affect the interaction of FEZ1 with SCOC. RF or ULK1 siRNA‐treated cells were co‐transfected with FEZ1-GFP and FLAG-SCOC, then incubated and immunoprecipitated as in (C). Western blot of lysates for ULK1, GFP and FLAG, and immunoprecipitates with GFP and FLAG antibodies.


( F ) siRNA depletion of SCOC reduces FEZ1 - ULK1 . HEK293 treated with either SCOC or ULK1 siRNAs were transfected with FEZ1 - GFP . FEZ1 - GFP was detected with anti ‐ GFP in both BN - PAGE SDS - PAGE . Endogenous ULK1 was detected with anti ‐ ULK1 antibody . Arrowheads indicate a FEZ1 - GFP endogenous ULK1 at 200 kDa a larger MW > 1236 kDa also detected in ( D ) in triple transfection . 200 KDa is sensitive to loss of SCOC ULK1 .

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(F) siRNA depletion of SCOC reduces FEZ1-ULK1 complex. HEK293 cells treated with either SCOC or ULK1 siRNAs were transfected with FEZ1-GFP. FEZ1-GFP was detected with anti‐GFP in both BN-PAGE and SDS-PAGE. Endogenous ULK1 was detected with anti‐ULK1 antibody. Arrowheads indicate a FEZ1-GFP and endogenous ULK1 complex at 200 kDa and a larger MW complex >1236 kDa also detected in (D) in the triple transfection. The 200 KDa complex is sensitive to loss of SCOC and ULK1.


( G ) SCOC can reduce interaction of ULK1 with MBP - FEZ1 . Immobilised MBP - FEZ1 was incubated with in vitro 35S ‐ methionine ‐ labelled Myc - ULK1 . Addition of increasing amounts of in vitro translated 35S ‐ methionine SCOC reduced amount of Myc - ULK1 bound . Top bottom panels are as described in ( A ) .

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(G) SCOC can reduce the interaction of ULK1 with MBP-FEZ1. Immobilised MBP-FEZ1 was incubated with in vitro 35S‐methionine‐labelled Myc-ULK1. Addition of increasing amounts of in vitro translated 35S‐methionine SCOC reduced the amount of Myc-ULK1 bound. Top and bottom panels are as described in (A).


( A ) SCOC interacts with UVRAG . HEK293 were transfected with FLAG - SCOC Myc - UVRAG , lysed incubated with anti ‐ FLAG ( left ) or anti ‐ Myc ( right ) antibodies . Co ‐ immunoprecipitation of Myc - UVRAG ( left ) FLAG - SCOC ( right ) was confirmed using anti ‐ Myc anti ‐ FLAG antibodies for western blotting . Asterisk in ( A - C ) ( D ) indicates non ‐ specific bands .

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(A) SCOC interacts with UVRAG. HEK293 cells were transfected with FLAG-SCOC and Myc-UVRAG, lysed and incubated with anti‐FLAG (left) or anti‐Myc (right) antibodies. Co‐immunoprecipitation of Myc-UVRAG (left) and FLAG-SCOC (right) was confirmed using anti‐Myc and anti‐FLAG antibodies for western blotting. Asterisk in (A-C) and (D) indicates non‐specific bands.


( B ) Interaction of SCOC with UVRAG is unaffected by FEZ1 - GFP . As in ( A ) , co ‐ immunoprecipitation was preformed in HEK293 transfected with FLAG - SCOC , Myc - UVRAG FEZ1 - GFP . Myc - UVRAG was detected as in ( A ) , FEZ1 - GFP was detected with anti ‐ GFP antibody .

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(B) Interaction of SCOC with UVRAG is unaffected by FEZ1-GFP. As in (A), co‐immunoprecipitation was preformed in HEK293 cells transfected with FLAG-SCOC, Myc-UVRAG and FEZ1-GFP. Myc-UVRAG was detected as in (A), FEZ1-GFP was detected with anti‐GFP antibody.