Quantification of EdU positive CMs ( mono - or multi - nucleated ) in vehicle and navitoclax treated animals . Data are mean ± S . E . M of n = 5 - 6 mice per group .

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Quantification of EdU positive CMs (mono- or multi-nucleated) in vehicle and navitoclax treated animals. Data are mean±S.E.M of n=5-6 mice per group.


% of Ki67 positive CMs in 29 month old INK - ATTAC mice treated with vehicle or AP20187 . Data are mean ± S . E . M of n = 4 - 6 mice per group .

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% of Ki67 positive CMs in 29 month old INK-ATTAC mice treated with vehicle or AP20187. Data are mean±S.E.M of n=4-6 mice per group.


Cloning and characterization of APG9 . A , Wild - type ( WT ; SEY6210 ) , apg9 ( THY154 ) , apg9Δ ( JKY007 ) , and the apg9Δ strain transformed with single copy ( CEN ; pAPG9 ( 414 ) ) or multicopy ( 2μ ; pAPG9 ( 424 ) ) plasmids encoding APG9 were grown to log phase in SMD . Protein extracts were prepared and analyzed by immunoblot using antiserum to API as described in Materials and Methods . The positions of precursor and mature API are indicated . The APG9 gene complements the prAPI accumulation phenotype of the apg9Δ mutant .

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Cloning and characterization of APG9. A, Wild-type (WT; SEY6210), apg9 (THY154), apg9Δ (JKY007), and the apg9Δ strain transformed with single copy (CEN; pAPG9(414)) or multicopy (2μ; pAPG9(424)) plasmids encoding APG9 were grown to log phase in SMD. Protein extracts were prepared and analyzed by immunoblot using antiserum to API as described in Materials and Methods. The positions of precursor and mature API are indicated. The APG9 gene complements the prAPI accumulation phenotype of the apg9Δ mutant.


B , WT ( SEY6210 ) , apg9Δ ( JKY007 ) , and the apg9Δ strain transformed with pAPG9 ( 414 ) centromeric plasmid were grown in SMD and transferred to SD ( − N ) as described in Materials and Methods . Aliquots were removed at the indicated times and spread onto YPD plates in triplicate . Numbers of viable colonies were determined after two to three days . The APG9 gene complements the starvation - sensitive phenotype of the apg9Δ mutant .

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B, WT (SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with pAPG9(414) centromeric plasmid were grown in SMD and transferred to SD(−N) as described in Materials and Methods. Aliquots were removed at the indicated times and spread onto YPD plates in triplicate. Numbers of viable colonies were determined after two to three days. The APG9 gene complements the starvation-sensitive phenotype of the apg9Δ mutant.


Apg9p is an unglycosylated , integral membrane protein . A , Wild - type ( WT ; SEY6210 ) , apg9Δ ( JKY007 ) , and the apg9Δ strain transformed with single copy ( CEN ) or multicopy ( 2μ ) plasmids encoding APG9 were grown to log phase in SMD . Protein extracts were prepared and analyzed by immunoblot using antiserum to Apg9p . Apg9p is detected as an ∼ 125 - kD protein .

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Apg9p is an unglycosylated, integral membrane protein. A, Wild-type (WT; SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with single copy (CEN) or multicopy (2μ) plasmids encoding APG9 were grown to log phase in SMD. Protein extracts were prepared and analyzed by immunoblot using antiserum to Apg9p. Apg9p is detected as an ∼125-kD protein.


. B , Hydrophobicity analysis of Apg9p based on the Kyte - Doolittle algorithm ( Kyte and Doolittle 1982 ) predicts six to eight transmembrane domains .

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. B, Hydrophobicity analysis of Apg9p based on the Kyte-Doolittle algorithm (Kyte and Doolittle 1982) predicts six to eight transmembrane domains.


C , Cells of wild - type strain YW5 - 1B were grown in YPD , converted to spheroplasts , and osmotically lysed as described in Materials and Methods . After a preclearing step to remove cell debris , the lysates were incubated with or without 1 % Triton X - 100 on ice for 30 min , as indicated . Centrifugation at 10 , 000 g for 1 h separated the lysate into supernatant ( S ) and pellet ( P ) fractions , which were analyzed by immunoblots with antiserum to Apg9p . Apg9p is found in the low - speed pellet in the absence of Triton X - 100 . Detergent extraction completely solubilizes Apg9p and causes it to appear in the supernatant

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C, Cells of wild-type strain YW5-1B were grown in YPD, converted to spheroplasts, and osmotically lysed as described in Materials and Methods. After a preclearing step to remove cell debris, the lysates were incubated with or without 1% Triton X-100 on ice for 30 min, as indicated. Centrifugation at 10,000 g for 1 h separated the lysate into supernatant (S) and pellet (P) fractions, which were analyzed by immunoblots with antiserum to Apg9p. Apg9p is found in the low-speed pellet in the absence of Triton X-100. Detergent extraction completely solubilizes Apg9p and causes it to appear in the supernatant fraction


D , Strain CTD1 harboring a multicopy 3 × HA APG9 plasmid was grown in YPD and converted to spheroplasts as described in Materials and Methods . A crude lysate was prepared and treated with or without endo H as indicated overnight at 37 ° C and analyzed by immunoblots with anti - HA antibodies or anti - CPY serum . Endo H treatment removes the N - linked glycosyl residues of CPY , causing a shift in its electrophoretic mobility . In contrast , Apg9p is not glycosylated and remains unchanged in the presence or absence of endo H treatment .

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D, Strain CTD1 harboring a multicopy 3×HA APG9 plasmid was grown in YPD and converted to spheroplasts as described in Materials and Methods. A crude lysate was prepared and treated with or without endo H as indicated overnight at 37°C and analyzed by immunoblots with anti-HA antibodies or anti-CPY serum. Endo H treatment removes the N-linked glycosyl residues of CPY, causing a shift in its electrophoretic mobility. In contrast, Apg9p is not glycosylated and remains unchanged in the presence or absence of endo H treatment.


Apg9p does not comigrate with typical endomembrane markers in sucrose density gradients . A and B , Strain STY1 ( pep4Δ ) was grown in YPD to log - phase , and osmotically lysed . Lysates were precleared , loaded on a sucrose density gradient ranging from 18 - 55 % ( wt / wt ) , and centrifuged for 2 . 5 h at 174 , 000 g as described in Materials and Methods . Fractions were collected from the top ( fraction number 1 ) to the bottom ( fraction number 16 ) . Fractions were subjected to SDS - PAGE and immunoblot with antiserum against Apg9p and : in A , Kex2p ( trans Golgi network ) , Pho8p ( vacuole ) , and Pma1p ( plasma membrane ) ; and in B , Sec12p ( ER ) and Pep12p ( endosome ) . The graphs in panels A and B are from the same gradient , but are presented separately for clarity .

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Apg9p does not comigrate with typical endomembrane markers in sucrose density gradients. A and B, Strain STY1 (pep4Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, loaded on a sucrose density gradient ranging from 18-55% (wt/wt), and centrifuged for 2.5 h at 174,000 g as described in Materials and Methods. Fractions were collected from the top (fraction number 1) to the bottom (fraction number 16). Fractions were subjected to SDS-PAGE and immunoblot with antiserum against Apg9p and: in A, Kex2p (trans Golgi network), Pho8p (vacuole), and Pma1p (plasma membrane); and in B, Sec12p (ER) and Pep12p (endosome). The graphs in panels A and B are from the same gradient, but are presented separately for clarity.


Apg9p does not comigrate with prAPI in a strain that accumulates Cvt / autophagic vesicles . Strain TK415 ( ypt7Δ ) was grown in YPD to log - phase , and osmotically lysed . Lysates were precleared , centrifuged at 100 , 000 g for 1 h , and the pellet fraction resuspended and centrifuged a second time . The resulting pellet fraction was subsequently loaded on a sucrose density gradient ranging from 18 - 55 % ( wt / wt ) , and centrifuged for 16 h at 174 , 000 g as described in Materials and Methods . 16 fractions were collected starting from the top and subjected to SDS - PAGE and immunoblot with antisera against Apg9p and API . The graph represents an average of two experiments .

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Apg9p does not comigrate with prAPI in a strain that accumulates Cvt/autophagic vesicles. Strain TK415 (ypt7Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, centrifuged at 100,000 g for 1 h, and the pellet fraction resuspended and centrifuged a second time. The resulting pellet fraction was subsequently loaded on a sucrose density gradient ranging from 18-55% (wt/wt), and centrifuged for 16 h at 174,000 g as described in Materials and Methods. 16 fractions were collected starting from the top and subjected to SDS-PAGE and immunoblot with antisera against Apg9p and API. The graph represents an average of two experiments.


Apg9p localizes to large perivacuolar punctate structures . Immunofluorescence microscopy of strains CTD1 ( apg9Δ ) , KSL12C ( vps4Δ ) , and SKD6 - 1D ( apg6Δ ) transformed with the multicopy plasmid 3 × HA APG9 . Cells were grown in YPD to log phase ( vegetative ) , and incubated further in SD ( − N ) medium with 1 mM PMSF for 3 h ( starvation ) . The cells were fixed with formaldehyde and examined by immunofluorescence microscopy as described in Materials and Methods . Anti - HA and FITC - conjugated antibodies mark Apg9p ( left ) , and the vacuole can be visualized by Nomarski optics ( middle ) . An overlay is shown in the right panels .

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Apg9p localizes to large perivacuolar punctate structures. Immunofluorescence microscopy of strains CTD1 (apg9Δ), KSL12C (vps4Δ), and SKD6-1D (apg6Δ) transformed with the multicopy plasmid 3×HA APG9. Cells were grown in YPD to log phase (vegetative), and incubated further in SD(−N) medium with 1 mM PMSF for 3 h (starvation). The cells were fixed with formaldehyde and examined by immunofluorescence microscopy as described in Materials and Methods. Anti-HA and FITC-conjugated antibodies mark Apg9p (left), and the vacuole can be visualized by Nomarski optics (middle). An overlay is shown in the right panels.


In vivo examination of the Apg9pGFP fusion protein . Wild - type ( SEY6210 ) cells expressing Apg9pGFP from a multicopy plasmid were grown in SMD to midlog phase and labeled with FM 4 - 64 to stain the vacuoles as described in Materials and Methods . The labeled cells were viewed directly with a Leica DM IRB confocal microscope . Large , punctate Apg9pGFP structures appear juxtaposed to the FM 4 - 64 - labeled vacuole membranes . In addition , smaller punctate structures appear throughout the cytoplasm .

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In vivo examination of the Apg9pGFP fusion protein. Wild-type (SEY6210) cells expressing Apg9pGFP from a multicopy plasmid were grown in SMD to midlog phase and labeled with FM 4-64 to stain the vacuoles as described in Materials and Methods. The labeled cells were viewed directly with a Leica DM IRB confocal microscope. Large, punctate Apg9pGFP structures appear juxtaposed to the FM 4-64-labeled vacuole membranes. In addition, smaller punctate structures appear throughout the cytoplasm.


Ultrastructural localization of Apg9p for immunoelectron microscopy . Strain STY1 ( pep4Δ ) expressing 3 × HA Apg9p was grown in YPD to log phase and transferred to SD ( − N ) medium for 2 h . The cells were fixed and stained with anti - HA antibody and 5 - nm colloidal gold - conjugated goat anti - mouse IgG as described in Materials and Methods . A , Section showing autophagic bodies in the vacuole lumen . B , Section showing a cytosolic autophagosome . Apg9p is seen in patches near the vacuole , but not on autophagic bodies or autophagosomes . AB , autophagic body ; AP , autophagosome ; N , nucleus ; V , vacuole ; VM , vacuolar membrane . Arrows point to areas of concentrated Apg9p .

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Ultrastructural localization of Apg9p for immunoelectron microscopy. Strain STY1 (pep4Δ) expressing 3×HA Apg9p was grown in YPD to log phase and transferred to SD(−N) medium for 2 h. The cells were fixed and stained with anti-HA antibody and 5-nm colloidal gold-conjugated goat anti-mouse IgG as described in Materials and Methods. A, Section showing autophagic bodies in the vacuole lumen. B, Section showing a cytosolic autophagosome. Apg9p is seen in patches near the vacuole, but not on autophagic bodies or autophagosomes. AB, autophagic body; AP, autophagosome; N, nucleus; V, vacuole; VM, vacuolar membrane. Arrows point to areas of concentrated Apg9p.


Vesicle accumulation test and kinetic analysis of prAPI import in the temperature conditional Apg9p mutant . A , Morphological analysis of apg9Δ indicates an import defect at an early step in the pathway . Wild - type ( YW5 - 1B ) and apg9Δ ( CTD1 ) cells were grown in YPD ( vegetative ) or transferred to SD ( − N ) ( starvation ) for 3 h in the presence of PMSF ( starvation ) and examined by DIC ( Nomarski ) microscopy ( Zeiss Axioplan ) . PMSF inhibits the degradation of autophagic bodies . Under starvation conditions , wild - type cells accumulate autophagic bodies when vesicle breakdown is blocked . However , under the same conditions , apg9Δ cells do not accumulate autophagic bodies , indicating that Apg9p is required for a step before vesicle fusion and release of the autophagic body into the vacuolar lumen . The apg9Δ strain transformed with the APG9 plasmid shows the same result as wild - type ( data not shown ) . B , The apg9ts strain is tightly blocked for prAPI import at nonpermissive temperature . Wild - type ( SEY6210 ) and apg9Δ ( JKY007 ) cells transformed with the apg9ts centromeric plasmid were incubated at 24 and 38 ° C for 5 min , pulse - labeled for 10 min , and then subjected to nonradioactive chase reactions for the indicated times . Samples at each time point were immunoprecipitated with antiserum to API and resolved by SDS - PAGE as described in Materials and Methods . API - immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager and the results are presented in the graph . The percent mature API was determined by dividing the mature API value over the sum of the precursor and mature API values for each time point .

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Vesicle accumulation test and kinetic analysis of prAPI import in the temperature conditional Apg9p mutant. A, Morphological analysis of apg9Δ indicates an import defect at an early step in the pathway. Wild-type (YW5-1B) and apg9Δ (CTD1) cells were grown in YPD (vegetative) or transferred to SD(−N) (starvation) for 3 h in the presence of PMSF (starvation) and examined by DIC (Nomarski) microscopy (Zeiss Axioplan). PMSF inhibits the degradation of autophagic bodies. Under starvation conditions, wild-type cells accumulate autophagic bodies when vesicle breakdown is blocked. However, under the same conditions, apg9Δ cells do not accumulate autophagic bodies, indicating that Apg9p is required for a step before vesicle fusion and release of the autophagic body into the vacuolar lumen. The apg9Δ strain transformed with the APG9 plasmid shows the same result as wild-type (data not shown). B, The apg9ts strain is tightly blocked for prAPI import at nonpermissive temperature. Wild-type (SEY6210) and apg9Δ (JKY007) cells transformed with the apg9ts centromeric plasmid were incubated at 24 and 38°C for 5 min, pulse-labeled for 10 min, and then subjected to nonradioactive chase reactions for the indicated times. Samples at each time point were immunoprecipitated with antiserum to API and resolved by SDS-PAGE as described in Materials and Methods. API-immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager and the results are presented in the graph. The percent mature API was determined by dividing the mature API value over the sum of the precursor and mature API values for each time point.


Analysis of the apg9ts strain indicates Apg9p is directly required for the vesicle formation step . A , The nature of prAPI binding and pelleting in the apg9ts strain . Spheroplasts of apg9ts were labeled for 10 min and chased for 30 min at 38 ° C . The spheroplasts were osmotically lysed in a buffer containing no salt ( 20 mM Pipes , pH 6 . 8 ) or a physiological concentration of salt ( 100 mM KOAc , 50 mM KCl , 5 mM MgCl2 , 20 mM Pipes , pH 6 . 8 ) and pelleted at 5 , 000 g . All samples were immunoprecipitated with antiserum to API and the cytosolic marker PGK . apg9ts retains the salt dependent binding and pelleting of prAPI . B , prAPI in the apg9ts strain accumulates in both a membrane - associated state and a large pelletable complex . Spheroplasts of apg9ts and ypt7Δ were labeled for 10 min and chased for 30 min at 38 ° C . The labeled spheroplasts were then osmotically lysed and separated into supernatant ( S ) and pellet ( P ) fractions by centrifugation at 5 , 000 g for 5 min . An aliquot was removed for the total lysate control ( T ) . The pellet fraction ( P ) was resuspended in 15 % Ficoll - 400 in gradient buffer ( 20 mM Pipes , 5 mM MgCl2 , complete EDTA - free protease inhibitor cocktail ) in the presence or absence of Triton X - 100 and overlaid with 13 and 2 % Ficoll - 400 in gradient buffer . The step gradients were centrifuged at 13 , 000 g for 10 min . Membrane - containing float ( F ) , nonfloat ( NF ) , and pellet ( P2 ) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods . The position of prAPI is indicated . C , prAPI in the apg9ts strain is protease accessible . Spheroplasts isolated from apg9ts and ypt7Δ cells were pulse - labeled for 10 min and chased for 30 min at 38 ° C . The labeled spheroplasts were then osmotically lysed and separated into low - speed supernatant ( S ) and pellet ( P ) fractions after a 5 , 000 g centrifugation step . The pellet fractions were subjected to protease treatment in the absence or presence of 0 . 2 % Triton X - 100 as described in Materials and Methods . The resulting samples were immunoprecipitated with antiserum to API and PGK . The immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager . The percent protease - protected prAPI was determined by dividing the value for prAPI in the protease - treated sample by the value for total prAPI before protease treatment . The percent PGK was determined by dividing the supernatant ( S ) or pellet ( P ) value over the sum of the S and P values .

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Analysis of the apg9ts strain indicates Apg9p is directly required for the vesicle formation step. A, The nature of prAPI binding and pelleting in the apg9ts strain. Spheroplasts of apg9ts were labeled for 10 min and chased for 30 min at 38°C. The spheroplasts were osmotically lysed in a buffer containing no salt (20 mM Pipes, pH 6.8) or a physiological concentration of salt (100 mM KOAc, 50 mM KCl, 5 mM MgCl2, 20 mM Pipes, pH 6.8) and pelleted at 5,000 g. All samples were immunoprecipitated with antiserum to API and the cytosolic marker PGK. apg9ts retains the salt dependent binding and pelleting of prAPI. B, prAPI in the apg9ts strain accumulates in both a membrane-associated state and a large pelletable complex. Spheroplasts of apg9ts and ypt7Δ were labeled for 10 min and chased for 30 min at 38°C. The labeled spheroplasts were then osmotically lysed and separated into supernatant (S) and pellet (P) fractions by centrifugation at 5,000 g for 5 min. An aliquot was removed for the total lysate control (T). The pellet fraction (P) was resuspended in 15% Ficoll-400 in gradient buffer (20 mM Pipes, 5 mM MgCl2, complete EDTA-free protease inhibitor cocktail) in the presence or absence of Triton X-100 and overlaid with 13 and 2% Ficoll-400 in gradient buffer. The step gradients were centrifuged at 13,000 g for 10 min. Membrane-containing float (F), nonfloat (NF), and pellet (P2) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods. The position of prAPI is indicated. C, prAPI in the apg9ts strain is protease accessible. Spheroplasts isolated from apg9ts and ypt7Δ cells were pulse-labeled for 10 min and chased for 30 min at 38°C. The labeled spheroplasts were then osmotically lysed and separated into low-speed supernatant (S) and pellet (P) fractions after a 5,000 g centrifugation step. The pellet fractions were subjected to protease treatment in the absence or presence of 0.2% Triton X-100 as described in Materials and Methods. The resulting samples were immunoprecipitated with antiserum to API and PGK. The immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager. The percent protease-protected prAPI was determined by dividing the value for prAPI in the protease-treated sample by the value for total prAPI before protease treatment. The percent PGK was determined by dividing the supernatant (S) or pellet (P) value over the sum of the S and P values.


( b ) Volcano plots of up ( red ) and down ( blue ) regulated genes ( compared to healthy control samples ) in S . Typhi and S . Paratyphi A positive individuals ( Nepal and Oxford ) . Black numbers indicate the up - and down - regulated genes compared to healthy controls .

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(b) Volcano plots of up (red) and down (blue) regulated genes (compared to healthy control samples) in S. Typhi and S. Paratyphi A positive individuals (Nepal and Oxford). Black numbers indicate the up- and down-regulated genes compared to healthy controls.


( d - e ) Scatter plots of BTMs enriched ( p > 0 . 05 ) in blood - culture positive samples in Nepal ( y - axis ) versus Oxford ( x - axis ) for typhoid fever ( d ) and paratyphoid fever ( e ) . For further details on BTMs refer to reference ( Chaussabel et al . , 2014 ) .

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(d-e) Scatter plots of BTMs enriched (p>0.05) in blood-culture positive samples in Nepal (y-axis) versus Oxford (x-axis) for typhoid fever (d) and paratyphoid fever (e). For further details on BTMs refer to reference (Chaussabel et al., 2014).


( a ) Single - sample GSEA Normalised Enrichment Scores ( NES ) of IFN and DC BTMs of individuals with blood - culture confirmed enteric fever in Nepal and Oxford .

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(a) Single-sample GSEA Normalised Enrichment Scores (NES) of IFN and DC BTMs of individuals with blood-culture confirmed enteric fever in Nepal and Oxford.


( b ) Heatmap of the 500 most variably expressed genes in samples of the Nepali cohort . Bar graph on top of the heatmap shows temperature of each individual at the time of sampling . Three samples labelled as ' 03NP - CONT ' are samples that grew bacterial contaminants and were thus excluded from the entire analysis .

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(b) Heatmap of the 500 most variably expressed genes in samples of the Nepali cohort. Bar graph on top of the heatmap shows temperature of each individual at the time of sampling. Three samples labelled as '03NP-CONT' are samples that grew bacterial contaminants and were thus excluded from the entire analysis.


( d ) Combined expression score for samples based on the 5 - gene signature for samples in the discovery cohort ( top ) and validation cohort ( bottom ) . Ox . CTRL : Oxford controls ( D0 ) ; CTRL : Nepali control samples . PTB : pulmonary TB ; DENV : Dengue samples ; bsPf : blood - stage P . falciparum ; SPT : S . Paratyphi A ; ST : S . Typhi . ST and SPT samples are derived from the challenge models as well as from Nepal .

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(d) Combined expression score for samples based on the 5-gene signature for samples in the discovery cohort (top) and validation cohort (bottom). Ox.CTRL: Oxford controls (D0); CTRL: Nepali control samples. PTB: pulmonary TB; DENV: Dengue samples; bsPf: blood-stage P. falciparum; SPT: S. Paratyphi A; ST: S. Typhi. ST and SPT samples are derived from the challenge models as well as from Nepal.


( b ) Dot plot of prediction probability of being class EF versus the expression score calculated on the bases of the 5 - gene signature ( based on gene array data ) .

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(b) Dot plot of prediction probability of being class EF versus the expression score calculated on the bases of the 5-gene signature (based on gene array data).


( c ) qPCR gene expression scores of the 5 - gene signature ( ∆ ∆ CT over PPIA ) for CTRLs , 03NP - sEF , 03NP - SPT and 03NP - ST samples from Nepal . Yellow diamonds in the 03NP - sEF category represent the 9 patients classified as EF based on the random forest algorithm .

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(c) qPCR gene expression scores of the 5-gene signature (∆∆CT over PPIA) for CTRLs, 03NP-sEF, 03NP-SPT and 03NP-ST samples from Nepal. Yellow diamonds in the 03NP-sEF category represent the 9 patients classified as EF based on the random forest algorithm.


( d ) qPCR expression values ( ∆ ∆ Ct over PPIA ) of the 5 - gene signature in control samples ( Oxford and Nepal ) , S . Paratyphi A ( 03NP - SPT ) or S . Typhi ( 03NP - ST ) in Nepal , samples at day 7 after challenge of participants who stayed well following challenge with S . Typhi ( nD7 ) , or typhoid diagnosis after challenge ( TD ) in the Vi - TCV

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(d) qPCR expression values (∆∆Ct over PPIA) of the 5-gene signature in control samples (Oxford and Nepal), S. Paratyphi A (03NP-SPT) or S. Typhi (03NP-ST) in Nepal, samples at day 7 after challenge of participants who stayed well following challenge with S. Typhi (nD7), or typhoid diagnosis after challenge (TD) in the Vi-TCV study


( e ) Combined qPCR expression score of the 5 - gene signature . Black arrows indicate outlier samples .

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(e) Combined qPCR expression score of the 5-gene signature. Black arrows indicate outlier samples.


( f ) Temperature and CRP for samples of which data was available ( CRP was only measured in the Oxford CHIM ) . D0 = pre - challenge baseline Vi - TCV study ; nD7 = day 7 samples of participants who stayed well following challenge ( Vi - TCV study ) ; SPT = S . Paratyphi A ( 03NP ) ; ST = S . Typhi ( 03NP ) ; TD = Typhoid diagnosis ( Vi - TCV study ) . ( g ) Spearman ' s rank correlation of the 5 - gene combined expression score and ( left ) temperature ( only nD7 and TD samples from the Oxford CHIM - Vi - TCV and SPT and ST cases from Nepal at presentation to hospital were included ) and ( right ) CRP ( CRP was only available for Oxford CHIM - Vi - TCV samples and we excluded D0 baseline measures ) .

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(f) Temperature and CRP for samples of which data was available (CRP was only measured in the Oxford CHIM). D0 = pre-challenge baseline Vi-TCV study; nD7 = day 7 samples of participants who stayed well following challenge (Vi-TCV study); SPT = S. Paratyphi A (03NP); ST = S. Typhi (03NP); TD = Typhoid diagnosis (Vi-TCV study). (g) Spearman's rank correlation of the 5-gene combined expression score and (left) temperature (only nD7 and TD samples from the Oxford CHIM - Vi-TCV and SPT and ST cases from Nepal at presentation to hospital were included) and (right) CRP (CRP was only available for Oxford CHIM - Vi-TCV samples and we excluded D0 baseline measures).


A . NLK but not kinase - negative NLK ( Flag - NLK - KM ; K155M ) caused mobility shift of EGFP - YAP . EGFP - YAP was transfected with empty vector , Flag - NLK - WT , or Flag - NLK - KM into HEK293T cells . Mobility shift was shown by immunoblotting .

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

A. NLK but not kinase-negative NLK (Flag-NLK-KM; K155M) caused mobility shift of EGFP-YAP. EGFP-YAP was transfected with empty vector, Flag-NLK-WT, or Flag-NLK-KM into HEK293T cells. Mobility shift was shown by immunoblotting.


B . NLK phosphorylates YAP in vitro . Flag - NLK - WT , or Flag - NLK - KM was immunoprecipitated with anti - Flag antibody from cell lysates of HEK293T cells transfected with each plasmid and subjected to in vitro kinase assay with bacterially purified GST - YAP in the presence of [ 32P - ATP ] . Phosphorylation of GST - YAP and autophosphorylation of NLK were examined by autoradiography ( top panel ) , and Coomassie Blue staining showed similar levels of GST - YAP and Flag - NLK ( bottom panel ) .

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

B. NLK phosphorylates YAP in vitro. Flag-NLK-WT, or Flag-NLK-KM was immunoprecipitated with anti-Flag antibody from cell lysates of HEK293T cells transfected with each plasmid and subjected to in vitro kinase assay with bacterially purified GST-YAP in the presence of [32P-ATP]. Phosphorylation of GST-YAP and autophosphorylation of NLK were examined by autoradiography (top panel), and Coomassie Blue staining showed similar levels of GST-YAP and Flag-NLK (bottom panel).


C . NLK interacts with YAP at the endogenous level . Immunoprecipitates from HEK293T cell lysates with indicated antibody were blotted with anti - YAP or anti - NLK antibody .

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C. NLK interacts with YAP at the endogenous level. Immunoprecipitates from HEK293T cell lysates with indicated antibody were blotted with anti-YAP or anti-NLK antibody.


B . NLK - WT but not NLK - KM reduces Ser127 phosphorylation of endogenous YAP . Cell lysates from HEK293 cells transfected with empty vector ( - ) , Flag - NLK - WT , or Flag - NLK - KM were immunoblotted with antibodies shown in the figure ( left panel ) . The ratio of p - YAP ( S127 ) or p - YAP ( S397 ) / total - YAP of three independent western blots was quantified ( right panel ) . Data are presented as mean ± sem . * * P < 0 . 01 . Student t - test was used for statistical analysis .

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

B. NLK-WT but not NLK-KM reduces Ser127 phosphorylation of endogenous YAP. Cell lysates from HEK293 cells transfected with empty vector (-), Flag-NLK-WT, or Flag-NLK-KM were immunoblotted with antibodies shown in the figure (left panel). The ratio of p-YAP(S127) or p-YAP(S397)/total-YAP of three independent western blots was quantified (right panel). Data are presented as mean ± sem. **P < 0.01. Student t-test was used for statistical analysis.


C . Deficiency of NLK leads to increased YAP phosphorylation at Ser127 . Lysates from wild type and two independent NLK knockout HEK293 cells were immunoblotted with antibodies indicated in the figure ( left panel ) . The ratio of p - YAP ( S127 ) or p - YAP ( S397 ) / total - YAP of three independent western blots was quantified ( right panel ) . Data are presented as mean ± sem . * P < 0 . 05 and * * P < 0 . 01 . Student t - test was used for statistical analysis .

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

C. Deficiency of NLK leads to increased YAP phosphorylation at Ser127. Lysates from wild type and two independent NLK knockout HEK293 cells were immunoblotted with antibodies indicated in the figure (left panel). The ratio of p-YAP(S127) or p-YAP(S397)/total-YAP of three independent western blots was quantified (right panel). Data are presented as mean ± sem. *P < 0.05 and **P < 0.01. Student t-test was used for statistical analysis.


D . Ectopic expression of NLK attenuates YAP phosphorylation at Ser127 while increases phosphorylation at Ser128 . Cell lysates from HEK293 cells transfected with empty vector ( - ) , Flag - NLK - WT , or Flag - NLK - KM were immunoblotted with antibodies shown in the figure .

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D. Ectopic expression of NLK attenuates YAP phosphorylation at Ser127 while increases phosphorylation at Ser128. Cell lysates from HEK293 cells transfected with empty vector (-), Flag-NLK-WT, or Flag-NLK-KM were immunoblotted with antibodies shown in the figure.


E . Substitution of Ser127 with alanine enhances phosphorylation at Ser128 , and vice versa . Cell lysates from HEK293T cells transfected with empty vector ( - ) , EGFP - YAP , EGFP - YAP - S127A , or EGFP - YAP - S128A were immunoblotted with antibodies shown in the figure .

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E. Substitution of Ser127 with alanine enhances phosphorylation at Ser128, and vice versa. Cell lysates from HEK293T cells transfected with empty vector (-), EGFP-YAP, EGFP-YAP-S127A, or EGFP-YAP-S128A were immunoblotted with antibodies shown in the figure.


A . Ectopic expression of NLK attenuates interaction between YAP and 14 - 3 - 3 . Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti - HA antibody and immunoblotted with anti - GFP antibody ( top two panels ) . Whole cell lysates ( WCL ) were immunoblotted with antibodies shown in the figure ( bottom three panels ) .

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A. Ectopic expression of NLK attenuates interaction between YAP and 14-3-3. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-HA antibody and immunoblotted with anti-GFP antibody (top two panels). Whole cell lysates (WCL) were immunoblotted with antibodies shown in the figure (bottom three panels).


B . Deficiency of NLK leads to increased interaction between YAP and 14 - 3 - 3 . Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti - YAP antibody and immunoblotted with anti - 14 - 3 - 3 antibody ( top two panels ) . WCL were immunoblotted with antibodies indicated in the figure ( bottom three panels ) .

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B. Deficiency of NLK leads to increased interaction between YAP and 14-3-3. Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti-YAP antibody and immunoblotted with anti-14-3-3 antibody (top two panels). WCL were immunoblotted with antibodies indicated in the figure (bottom three panels).


C . Phosphorylation status of Ser127 or Ser128 determines interaction between 14 - 3 - 3 and YAP . Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti - Flag antibody and immunoblotted with anti - HA antibodies ( top two panels ) . Lysates were immunoblotted with anti - HA or anti - Flag antibodies ( bottom two panels ) . " n . s . " arrow indicates a non - specific band also visible in the non - transfected lane .

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C. Phosphorylation status of Ser127 or Ser128 determines interaction between 14-3-3 and YAP. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-HA antibodies (top two panels). Lysates were immunoblotted with anti-HA or anti-Flag antibodies (bottom two panels). "n.s." arrow indicates a non-specific band also visible in the non-transfected lane.


D . Phosphorylation status of Ser127 or Ser128 determines subcellular localization of YAP . Immunofluorescence analysis was performed in HeLa cells transfected with EGFP - YAP , EGFP - YAP - S127A , EGFP - YAP - S128A , or EGFP - YAP - S128D at high cell density . Figure shows a representative of multiple areas . Nuclei were stained with Hoechst 33342 . Scale bars : 20 μm .

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D. Phosphorylation status of Ser127 or Ser128 determines subcellular localization of YAP. Immunofluorescence analysis was performed in HeLa cells transfected with EGFP-YAP, EGFP-YAP-S127A, EGFP-YAP-S128A, or EGFP-YAP-S128D at high cell density. Figure shows a representative of multiple areas. Nuclei were stained with Hoechst 33342. Scale bars: 20 μm.


E . NLK knockout leads to increased cytoplasmic localization of endogenous YAP . Immunoblot analysis shows significant reduction of NLK level in NLK knockout HEK293 cell pools ( top panel ) .

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E. NLK knockout leads to increased cytoplasmic localization of endogenous YAP. Immunoblot analysis shows significant reduction of NLK level in NLK knockout HEK293 cell pools (top panel).


E . NLK knockout leads to increased cytoplasmic localization of endogenous YAP . To determine subcellular localization of YAP indirect immunofluorescence analysis was performed in NLK knockout HEK293 cell pools ( bottom panel ) . Figure shows a representative of multiple areas . Nuclei were stained with DAPI . Scale bars : 20 μm .

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E. NLK knockout leads to increased cytoplasmic localization of endogenous YAP. To determine subcellular localization of YAP indirect immunofluorescence analysis was performed in NLK knockout HEK293 cell pools (bottom panel). Figure shows a representative of multiple areas. Nuclei were stained with DAPI. Scale bars: 20 μm.


A . Localization of endogenous YAP and NLK in low ( LD ) or high density ( HD ) MCF10A ( upper panel ) or NIH3T3 cells ( bottom panel ) was examined by indirect immunofluorescence . Nuclei were stained with DAPI . Scale bars : 20 μm .

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A. Localization of endogenous YAP and NLK in low (LD) or high density (HD) MCF10A (upper panel) or NIH3T3 cells (bottom panel) was examined by indirect immunofluorescence. Nuclei were stained with DAPI. Scale bars: 20 μm.


B . Level of NLK decreases in a cell density - dependent manner . Lysates from low ( LD ) and high density ( HD ) of MCF10A , HEK293T , and NIH3T3 cells were immunoblotted with antibodies indicated in the figure .

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B. Level of NLK decreases in a cell density-dependent manner. Lysates from low (LD) and high density (HD) of MCF10A, HEK293T, and NIH3T3 cells were immunoblotted with antibodies indicated in the figure.


C . Level of endogenous NLK mRNA is unaffected , whereas the expression of YAP target genes was reduced , at high cell density . Quantitative real - time PCR analyses for expression of AMOTL2 , CTGF , CYR61 , and NLK in low ( LD ) and high density ( HD ) NIH3T3 cells were performed . Quantification of AMOTL2 , CTGF , CYR61 , and NLK mRNA was normalized with the level of Gapdh . Data represent average values from a representative of multiple experiments performed in triplicate . Error bars indicate standard deviations of triplicate measurements . Data are presented as mean ± sem . * P < 0 . 05 and * * * P < 0 . 005 . Student t - test was used for statistical analysis .

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C. Level of endogenous NLK mRNA is unaffected, whereas the expression of YAP target genes was reduced, at high cell density. Quantitative real-time PCR analyses for expression of AMOTL2, CTGF, CYR61, and NLK in low (LD) and high density (HD) NIH3T3 cells were performed. Quantification of AMOTL2, CTGF, CYR61, and NLK mRNA was normalized with the level of Gapdh. Data represent average values from a representative of multiple experiments performed in triplicate. Error bars indicate standard deviations of triplicate measurements. Data are presented as mean ± sem. *P < 0.05 and ***P < 0.005. Student t-test was used for statistical analysis.


A . Knockout of NLK reduces the interaction between endogenous YAP and TEAD . Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti - TEAD1 antibody and immunoblotted with anti - TEAD1 antibody or anti - YAP antibody ( top panel ) . Whole cell lysates ( WCL ) were immunoblotted with antibodies indicated in the figure .

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A. Knockout of NLK reduces the interaction between endogenous YAP and TEAD. Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti-TEAD1 antibody and immunoblotted with anti-TEAD1 antibody or anti-YAP antibody (top panel). Whole cell lysates (WCL) were immunoblotted with antibodies indicated in the figure.


B . Phosphorylation status of Ser127 or Ser128 determines interaction between TEAD1 and YAP . Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti - Flag antibody and immunoblotted with indicated antibodies in the figure .

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B. Phosphorylation status of Ser127 or Ser128 determines interaction between TEAD1 and YAP. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies in the figure.


C . Downregulation of nemo with RNAi achieved in the posterior compartment of wing imaginal discs ( co - expression of GFP serves to mark the posterior compartment ) using the hh - Gal4 driver results in decreased expression of the Yorkie target gene DIAP1 . Two different RNAi constructs produce similar effect . Panels below show Z - stacks of magnified areas around the A / P border ( left ) . Quantification of DIAP1 levels achieved upon posterior - downregulation of nemo , measured as normalized posterior - to - anterior levels and compared to similar expression of GFP . Data are present as mean ± sem , n = 8 discs . * P < 0 . 05 . Student t - test was used for statistical analysis ( right ) . Scale bars : 50 µm .

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C. Downregulation of nemo with RNAi achieved in the posterior compartment of wing imaginal discs (co-expression of GFP serves to mark the posterior compartment) using the hh-Gal4 driver results in decreased expression of the Yorkie target gene DIAP1. Two different RNAi constructs produce similar effect. Panels below show Z-stacks of magnified areas around the A/P border (left). Quantification of DIAP1 levels achieved upon posterior-downregulation of nemo, measured as normalized posterior-to-anterior levels and compared to similar expression of GFP. Data are present as mean ± sem, n=8 discs. *P < 0.05. Student t-test was used for statistical analysis (right). Scale bars: 50 µm.


D . Yorkie is epistatic to Nemo : co - overexpression of the constitutively active mutant yki [ S168A ] together with nmo - RNAi produces typical yki [ S168A ] phenotypes : severe overgrowth of the affected area with concomitant increase in yki target gene expression ( DIAP1 ) . Due to lethality of the hh - Gal4 - driven co - overexpression , another posterior driver en - Gal4 was used instead ; anti - Hh staining ( green ) was used to mark the posterior compartment ( left panel ) . Quantification posterior DIAP1 levels in UAS - RNAi - nemo ; en - Gal4 and UAS - yki [ S168A ] ; en - Gal4 , presented as in ( C ) ; n = 3 - 6 discs . Posterior DIAP1 levels in UAS - RNAi - nemo ; en - Gal4 are also significantly different from control UAS - GFP ; en - Gal4 discs ( right panel ) . Data are presented as mean ± sem . * * * P < 0 . 005 . Student t - test was used for statistical analysis ( right ) . Scale bars : 50 µm .

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D. Yorkie is epistatic to Nemo: co-overexpression of the constitutively active mutant yki[S168A] together with nmo-RNAi produces typical yki[S168A] phenotypes: severe overgrowth of the affected area with concomitant increase in yki target gene expression (DIAP1). Due to lethality of the hh-Gal4-driven co-overexpression, another posterior driver en-Gal4 was used instead; anti-Hh staining (green) was used to mark the posterior compartment (left panel). Quantification posterior DIAP1 levels in UAS-RNAi-nemo; en-Gal4 and UAS-yki[S168A]; en-Gal4, presented as in (C); n= 3-6 discs. Posterior DIAP1 levels in UAS-RNAi-nemo; en-Gal4 are also significantly different from control UAS-GFP; en-Gal4 discs (right panel). Data are presented as mean ± sem. ***P < 0.005. Student t-test was used for statistical analysis (right). Scale bars: 50 µm.


E . Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout . HEK293 wild type or HEK293 NLK knockout cells were transfected with indicated plasmids ( " EV " stands for " empty vector " ) , and the wounded areas after 0 and 24 hr were measured by using the TScratch program ( left panel ) . Graphs show percentage of unfilled areas at 24 hr compared to 0 hr , and average values from a representative of multiple experiments performed in triplicate ( right - top panel ) . Lysates were immunoblotted with antibodies indicated in the figure ( right - bottom panel ) . Error bars indicate standard deviations of triplicate measurements . Data are presented as mean ± sem . * P < 0 . 05 . Student t - test was used for statistical analysis ( right ) . Scale bars : 200 μm .

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

E. Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout. HEK293 wild type or HEK293 NLK knockout cells were transfected with indicated plasmids ("EV" stands for "empty vector"), and the wounded areas after 0 and 24 hr were measured by using the TScratch program (left panel). Graphs show percentage of unfilled areas at 24 hr compared to 0 hr, and average values from a representative of multiple experiments performed in triplicate (right-top panel). Lysates were immunoblotted with antibodies indicated in the figure (right-bottom panel). Error bars indicate standard deviations of triplicate measurements. Data are presented as mean ± sem. *P < 0.05. Student t-test was used for statistical analysis (right). Scale bars: 200 μm.


E . Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout . HEK293 wild type or HEK293 NLK knockout cells were transfected with indicated plasmids ( " EV " stands for " empty vector " ) , and the wounded areas after 0 and 24 hr were measured by using the TScratch program ( left panel ) . Graphs show percentage of unfilled areas at 24 hr compared to 0 hr , and average values from a representative of multiple experiments performed in triplicate ( right - top panel ) .

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E. Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout. HEK293 wild type or HEK293 NLK knockout cells were transfected with indicated plasmids ("EV" stands for "empty vector"), and the wounded areas after 0 and 24 hr were measured by using the TScratch program (left panel). Graphs show percentage of unfilled areas at 24 hr compared to 0 hr, and average values from a representative of multiple experiments performed in triplicate (right-top panel).


Immunoblot analysis of ADAR1 p110 , ADAR1 p150 and ADAR2 expression in various organs isolated from wild - type ( WT ) mice . The expression of GAPDH is shown as a reference .

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Immunoblot analysis of ADAR1 p110, ADAR1 p150 and ADAR2 expression in various organs isolated from wild-type (WT) mice. The expression of GAPDH is shown as a reference.


The relative expression of ADAR1 p150 mRNA ( B ) and total ADAR1 mRNA ( C ) in sorted DN , DP , 4SP and 8SP thymocytes isolated from WT mice . The relative expression of ADAR1 p150 mRNA ( B ) and total ADAR1 mRNA ( C ) were normalized to the level of expression in DN thymocytes ( DN values set to 1 ) . Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM ( n = 5 ; Mann - Whitney U - test , * p < 0 . 05 , * * p < 0 . 01 ) .

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The relative expression of ADAR1 p150 mRNA (B) and total ADAR1 mRNA (C) in sorted DN, DP, 4SP and 8SP thymocytes isolated from WT mice. The relative expression of ADAR1 p150 mRNA (B) and total ADAR1 mRNA (C) were normalized to the level of expression in DN thymocytes (DN values set to 1). Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 5; Mann-Whitney U-test, *p < 0.05, **p < 0.01).


The relative expression of type I interferon - stimulated genes ( ISGs ) in sorted DN , DP , 4SP and 8SP thymocytes isolated from WT mice . Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , * p < 0 . 05 ) . The relative expression of ISGs was normalized against the level of expression in DN thymocytes ( DN values set to 1 ) .

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The relative expression of type I interferon-stimulated genes (ISGs) in sorted DN, DP, 4SP and 8SP thymocytes isolated from WT mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The relative expression of ISGs was normalized against the level of expression in DN thymocytes (DN values set to 1).


Total number of thymocytes in wild - type ( WT ) and CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice .

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Total number of thymocytes in wild-type (WT) and CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice.


Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - CD4 and anti - CD8 antibodies and analyzed by flow cytometry . Representative images are shown ( B ) .

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Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Representative images are shown (B).


The percentages of DN , DP , 4SP , and 8SP thymocytes were compared between WT and Cd4cre A1fl / fl mice ( C ) .

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The percentages of DN, DP, 4SP, and 8SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (C).


Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 and anti - TCRβ antibodies and were gated by the high expression of TCRβ ( TCRβhigh ) . Representative images are shown ( D ) .

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Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8 and anti-TCRβ antibodies and were gated by the high expression of TCRβ (TCRβhigh). Representative images are shown (D).


The percentages of DN , DP , 4SP , and 8SP thymocytes were compared between WT and Cd4cre A1fl / fl mice ( E ) .

The percentages of DN, DP, 4SP, and 8SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (E).


Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 , anti - CD69 and anti - MHC - I antibodies and were gated on 4SP thymocytes . Representative images are shown ( upper panel ) . The percentages of CD69 + MHC - I - ( SM ) , CD69 + MHC - I + ( M1 ) , and CD69 - MHC - I - ( M2 ) thymocytes were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) .

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Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8, anti-CD69 and anti-MHC-I antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD69+MHC-I- (SM), CD69+MHC-I+ (M1), and CD69-MHC-I- (M2) thymocytes were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with antibodies against CD5 and analyzed by flow cytometry . Representative flow cytometric images for DP and 4SP thymocytes are shown ( upper panel ) . The percentages of CD5 - DP and 4SP thymocytes were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) .

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Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with antibodies against CD5 and analyzed by flow cytometry. Representative flow cytometric images for DP and 4SP thymocytes are shown (upper panel). The percentages of CD5- DP and 4SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Thymocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 , anti - CD25 and anti - Foxp3 antibodies and were gated on 4SP thymocytes . Representative images are shown ( upper panel ) . The percentages of CD25 + Foxp3 + 4SP thymocytes ( thymic Treg cells ) were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) .

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Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8, anti-CD25 and anti-Foxp3 antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD25+Foxp3+ 4SP thymocytes (thymic Treg cells) were compared between WT and Cd4cre A1fl/fl mice (lower panel).


CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice were crossed with HY - TCR + mice to generate Cd4cre A1fl / fl HY - TCR + mice . Thymocytes isolated from male ( A ) and female ( B ) HY - TCR + or Cd4cre A1fl / fl HY - TCR + mice were stained with anti - CD4 and anti - CD8 antibodies and analyzed by flow cytometry . Representative images are shown ( left panels ) . The absolute number of DP and 8SP thymocytes were compared between HY - TCR + and Cd4cre A1fl / fl HY - TCR + mice ( right panels ) .

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CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice were crossed with HY-TCR+ mice to generate Cd4cre A1fl/fl HY-TCR+ mice. Thymocytes isolated from male (A) and female (B) HY-TCR+ or Cd4cre A1fl/fl HY-TCR+ mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Representative images are shown (left panels). The absolute number of DP and 8SP thymocytes were compared between HY-TCR+ and Cd4cre A1fl/fl HY-TCR+ mice (right panels).


Thymocytes isolated from WT and Cd4cre A1fl / fl mice were labeled with Cell Proliferation Dye and then stimulated with plate - bound anti - CD3 and anti - CD28 antibodies for 4 days . Apoptotic ( Annexin V + ) and proliferating thymocytes ( reduced intensity of Proliferation Dye ) were determined by flow cytometry . Representative flow cytometric images are shown ( left panel ) . The percentages of either Annexin V + or Annexin V - proliferating thymocytes were compared between WT ( n = 5 ) and Cd4cre A1fl / fl ( n = 4 ) mice ( right panel ) .

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

Thymocytes isolated from WT and Cd4cre A1fl/fl mice were labeled with Cell Proliferation Dye and then stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 4 days. Apoptotic (Annexin V+) and proliferating thymocytes (reduced intensity of Proliferation Dye) were determined by flow cytometry. Representative flow cytometric images are shown (left panel). The percentages of either Annexin V+ or Annexin V- proliferating thymocytes were compared between WT (n = 5) and Cd4cre A1fl/fl (n = 4) mice (right panel).


The relative expression of anti - apoptotic genes in sorted 4SP thymocytes isolated from WT and Cd4cre A1fl / fl mice . Values represent the relative gene expression normalized to Actinβ mRNA and are displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , * p < 0 . 05 , N . S . ; not significant ) . The mean value for the relative expression of each anti - apoptotic gene in WT 4SP thymocytes was set as one .

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The relative expression of anti-apoptotic genes in sorted 4SP thymocytes isolated from WT and Cd4cre A1fl/fl mice. Values represent the relative gene expression normalized to Actinβ mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05, N.S.; not significant). The mean value for the relative expression of each anti-apoptotic gene in WT 4SP thymocytes was set as one.


Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti - CD3 - stimulated thymocytes isolated from WT and Cd4cre A1fl / fl mice . The expression of GAPDH is shown as a reference . Representative images are shown ( left panel ) . The band intensity of p - ZAP70 and p - PLCγ1 was normalized to that of total ZAP70 and PLCγ1 ,

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Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti-CD3-stimulated thymocytes isolated from WT and Cd4cre A1fl/fl mice. The expression of GAPDH is shown as a reference. Representative images are shown (left panel). The band intensity of p-ZAP70 and p-PLCγ1 was normalized to that of total ZAP70 and PLCγ1, respectively


Incidence of spontaneous colitis observed in wild - type ( WT ) and CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice ( n = 11 for each group ;

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Incidence of spontaneous colitis observed in wild-type (WT) and CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice (n = 11 for each group;


Representative images of HE staining of the colon dissected from a WT and Cd4cre A1fl / fl mouse . Scale bar , 200 μm .

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Representative images of HE staining of the colon dissected from a WT and Cd4cre A1fl/fl mouse. Scale bar, 200 μm.


Lymphocytes isolated from the colonic lamina propria of WT and Cd4cre A1fl / fl mice were stained with anti - IFNγ , anti - IL17 or anti - Foxp3 antibodies together with anti - CD4 antibodies and analyzed by flow cytometry . Representative images are shown ( left panel ) . The absolute number of IFNγ - , IL17 - , or Foxp3 - expressing CD4 + T cells were compared between WT and Cd4cre A1fl / fl mice ( right panel ) .

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Lymphocytes isolated from the colonic lamina propria of WT and Cd4cre A1fl/fl mice were stained with anti-IFNγ, anti-IL17 or anti-Foxp3 antibodies together with anti-CD4 antibodies and analyzed by flow cytometry. Representative images are shown (left panel). The absolute number of IFNγ-, IL17-, or Foxp3-expressing CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (right panel).


Splenocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - IFNγ and anti - IL17 together with anti - CD4 antibodies and were gated on CD4 + cells . Representative flow cytometry images are shown ( upper panel ) . The percentages of IFNγ - and IL17 - secreting CD4 + T cells were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) .

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Splenocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-IFNγ and anti-IL17 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (upper panel). The percentages of IFNγ- and IL17-secreting CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Splenocytes isolated from WT and Cd4cre A1fl / fl mice were stained with anti - Foxp3 together with anti - CD4 antibodies and were gated on CD4 + cells . Representative flow cytometry images are shown ( upper panel ) . The percentages of Foxp3 - expressing CD4 + T cells were compared between WT and Cd4cre A1fl / fl mice ( lower panel ) .

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Splenocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-Foxp3 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (upper panel). The percentages of Foxp3-expressing CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Effector T ( Teff ) cells isolated from the spleen of a WT mouse were stimulated with soluble anti - CD3 ( 5 μg / ml ) in the presence of mitomycin C - treated splenocytes and were cultured with regulatory T ( Treg ) cells isolated from WT and Cd4cre A1fl / fl mice at the indicated ratios . After 72 h , the proliferative activity was measured . Values are displayed as the mean ± SEM ( n = 3 for each group ; Mann - Whitney U - test , N . S . ; not significant ) . The mean value for the suppression of proliferative activity of Teff cells in a condition without Treg cells was set as 0 % .

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Effector T (Teff) cells isolated from the spleen of a WT mouse were stimulated with soluble anti-CD3 (5 μg/ml) in the presence of mitomycin C-treated splenocytes and were cultured with regulatory T (Treg) cells isolated from WT and Cd4cre A1fl/fl mice at the indicated ratios. After 72 h, the proliferative activity was measured. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, N.S.; not significant). The mean value for the suppression of proliferative activity of Teff cells in a condition without Treg cells was set as 0%.


RNA editing in the 3 ′ UTR ( C ) and intronic region ( D ) of each gene indicated in the figures with their chromosomal location was validated by direct Sanger sequencing and the frequency of editing was compared among wild - type ( WT ) DP , WT 4SP thymocytes , and 4SP thymocytes isolated from CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice .

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RNA editing in the 3′UTR (C) and intronic region (D) of each gene indicated in the figures with their chromosomal location was validated by direct Sanger sequencing and the frequency of editing was compared among wild-type (WT) DP, WT 4SP thymocytes, and 4SP thymocytes isolated from CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice.


The relative expression of each interferon - stimulated gene ( ISG ) indicated in the figure in sorted DP and 4SP thymocytes isolated from WT and Cd4cre A1fl / fl mice . Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , * p < 0 . 05 ) . The mean value for the relative expression of each ISG in WT DP thymocytes was set as one .

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

The relative expression of each interferon-stimulated gene (ISG) indicated in the figure in sorted DP and 4SP thymocytes isolated from WT and Cd4cre A1fl/fl mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The mean value for the relative expression of each ISG in WT DP thymocytes was set as one.


Immunoblot analysis of total and phosphorylated STAT1 expression in thymocytes isolated from WT and Cd4cre A1fl / fl mice ( n = 2 for each group ) . The expression of GAPDH is shown as a reference .

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Immunoblot analysis of total and phosphorylated STAT1 expression in thymocytes isolated from WT and Cd4cre A1fl/fl mice (n = 2 for each group). The expression of GAPDH is shown as a reference.


CD4 + cell - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice were crossed with MDA5 knockout ( Ifih1 - / - ) mice to generate Cd4cre A1fl / fl Ifih1 - / - mice . Thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - CD4 , anti - CD8 and anti - TCRβ antibodies and analyzed by flow cytometry . Representative images are shown ( left panel ) . The percentage of DN , DP , 4SP and 8SP thymocytes were compared among Ifih1 - / - ( n = 4 ) , Cd4cre A1fl / fl Ifih1 - / - ( n = 5 ) and Cd4cre A1fl / fl ( n = 7 ) mice ( right panel ) . The same data shown in Fig 2C was used for Cd4cre A1fl / fl mice .

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CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice were crossed with MDA5 knockout (Ifih1-/-) mice to generate Cd4cre A1fl/fl Ifih1-/- mice. Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8 and anti-TCRβ antibodies and analyzed by flow cytometry. Representative images are shown (left panel). The percentage of DN, DP, 4SP and 8SP thymocytes were compared among Ifih1-/- (n = 4), Cd4cre A1fl/fl Ifih1-/- (n = 5) and Cd4cre A1fl/fl (n = 7) mice (right panel). The same data shown in Fig 2C was used for Cd4cre A1fl/fl mice.


The relative expression of each interferon - stimulated gene ( ISG ) indicated in the figure in sorted 4SP thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice . Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , * p < 0 . 05 ) . The mean value for the relative expression of each ISG in Ifih1 - / - 4SP thymocytes was set as one .

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

The relative expression of each interferon-stimulated gene (ISG) indicated in the figure in sorted 4SP thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The mean value for the relative expression of each ISG in Ifih1-/- 4SP thymocytes was set as one.


Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti - CD3 - stimulated thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice . The expression of GAPDH is shown as a reference . Representative images are shown ( left panel ) . The band intensity of p - ZAP70 and p - PLCγ1 was normalized to that of total ZAP70 and PLCγ1 , respectively and displayed as the mean ± SEM ( n = 3 ; Mann - Whitney U - test , N . S . ; not significant ) ( right panel ) by setting the mean value of stimulated Ifih1 - / - samples at 60 sec as 1 .

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Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti-CD3-stimulated thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice. The expression of GAPDH is shown as a reference. Representative images are shown (left panel). The band intensity of p-ZAP70 and p-PLCγ1 was normalized to that of total ZAP70 and PLCγ1, respectively and displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, N.S.; not significant) (right panel) by setting the mean value of stimulated Ifih1-/- samples at 60 sec as 1.


Thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stimulated with plate - bound anti - CD3 ( 5 μg / ml ) and anti - CD28 ( 1 μg / ml ) antibodies for 72 h and the proliferative activity was measured . Values are displayed as the mean ± SEM ( n = 3 for each group ; Mann - Whitney U - test , * p < 0 . 05 ) . The mean value for the activity of Ifih1 - / - thymocytes in an unstimulated condition was set as one . For comparative analysis , the same data shown in Appendix Fig S1B was used for Cd4cre A1fl / fl mice .

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Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stimulated with plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) antibodies for 72 h and the proliferative activity was measured. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, *p < 0.05). The mean value for the activity of Ifih1-/- thymocytes in an unstimulated condition was set as one. For comparative analysis, the same data shown in Appendix Fig S1B was used for Cd4cre A1fl/fl mice.


Thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - CD4 , anti - CD8 , anti - CD69 and anti - MHC - I antibodies and were gated on 4SP thymocytes . Representative images are shown ( upper panel ) . The percentages of CD69 + MHC - I - ( SM ) , CD69 + MHC - I + ( M1 ) , and CD69 - MHC - I - ( M2 ) thymocytes were compared among Ifih1 - / - ( n = 5 ) , Cd4cre A1fl / fl Ifih1 - / - ( n = 3 ) and Cd4cre A1fl / fl ( n = 3 ) mice ( lower panel ) . The same data shown in Fig 2F was used for Cd4cre A1fl / fl mice .

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Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8, anti-CD69 and anti-MHC-I antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD69+MHC-I- (SM), CD69+MHC-I+ (M1), and CD69-MHC-I- (M2) thymocytes were compared among Ifih1-/- (n = 5), Cd4cre A1fl/fl Ifih1-/- (n = 3) and Cd4cre A1fl/fl (n = 3) mice (lower panel). The same data shown in Fig 2F was used for Cd4cre A1fl/fl mice.


Thymocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - CD4 , anti - CD8 , anti - CD25 and anti - Foxp3 antibodies and were gated on 4SP thymocytes . Representative images are shown ( upper panel ) . The percentages of CD25 + Foxp3 + 4SP thymocytes ( thymic Treg cells ) were compared among Ifih1 - / - , Cd4cre A1fl / fl Ifih1 - / - and Cd4cre A1fl / fl mice ( lower panel ) . The same data shown in Fig 2H was used for Cd4cre A1fl / fl mice .

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Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8, anti-CD25 and anti-Foxp3 antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD25+Foxp3+ 4SP thymocytes (thymic Treg cells) were compared among Ifih1-/-, Cd4cre A1fl/fl Ifih1-/- and Cd4cre A1fl/fl mice (lower panel). The same data shown in Fig 2H was used for Cd4cre A1fl/fl mice.


Splenocytes isolated from Ifih1 - / - and Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - IFNγ and anti - IL17 together with anti - CD4 antibodies and were gated on CD4 + cells . Representative flow cytometry images are shown ( left panel ) . The percentages of IFNγ - and IL17 - secreting CD4 + T cells were compared among Ifih1 - / - ( n = 4 ) , Cd4cre A1fl / fl Ifih1 - / - ( n = 5 ) and Cd4cre A1fl / fl mice ( n = 6 ) ( right panel ) . The same data shown in Fig 4E was used for Cd4cre A1fl / fl mice .

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Splenocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-IFNγ and anti-IL17 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (left panel). The percentages of IFNγ- and IL17-secreting CD4+ T cells were compared among Ifih1-/- (n = 4), Cd4cre A1fl/fl Ifih1-/- (n = 5) and Cd4cre A1fl/fl mice (n = 6) (right panel). The same data shown in Fig 4E was used for Cd4cre A1fl/fl mice.


No incidence of spontaneous colitis observed in Ifih1 - / - ( n = 5 ) and Cd4cre A1fl / fl Ifih1 - / - ( n = 11 ) mice . For side - by - side comparison with Cd4cre A1fl / fl

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No incidence of spontaneous colitis observed in Ifih1-/- (n = 5) and Cd4cre A1fl/fl Ifih1-/- (n = 11) mice. For side-by-side comparison with Cd4cre A1fl/fl mice


Representative images of HE staining of colon dissected from an Ifih1 - / - and a Cd4cre A1fl / fl Ifih1 - / - mouse . Scale bar , 200 μm .

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Representative images of HE staining of colon dissected from an Ifih1-/- and a Cd4cre A1fl/fl Ifih1-/- mouse. Scale bar, 200 μm.


A Indicated tail - anchored ( TA ) proteins were in vitro translated in rabbit reticulocyte lysate ( RRL ) using RNAs containing a stop codon . Translations were carried out in the presence of 2 μM HisTrx or HisTrx - SGTA ( HT - SGTA ) , followed by incubation with HisPur Cobalt resin Bound proteins were eluted at high imidazole concentration , samples resolved by SDS - PAGE and results visualised by phosphorimaging . Sec61β 3R carries three Arg residues within Sec61β transmembrane domain ( TMD ) which abolish its hydrophobic character and was used as a negative control for SGTA binding . A schematic representation of a TA - protein is also shown . Open circles indicate TA - proteins selectively bound by HisTrx - SGTA . RAMP4 - stress - associated endoplasmic reticulum protein 1 ; Syb2 - synaptobrevin 2 ; Synt5 - syntaxin 5 . B As for ( A ) but total translation products ( input ) were resolved by SDS - PAGE and results visualised by phosphorimaging . When total products are analysed high amounts of haemoglobin in the RRL distort the migration of Sec61β and its variant .

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

A Indicated tail-anchored (TA) proteins were in vitro translated in rabbit reticulocyte lysate (RRL) using RNAs containing a stop codon. Translations were carried out in the presence of 2 μM HisTrx or HisTrx-SGTA (HT-SGTA), followed by incubation with HisPur Cobalt resin Bound proteins were eluted at high imidazole concentration, samples resolved by SDS-PAGE and results visualised by phosphorimaging. Sec61β 3R carries three Arg residues within Sec61β transmembrane domain (TMD) which abolish its hydrophobic character and was used as a negative control for SGTA binding. A schematic representation of a TA-protein is also shown. Open circles indicate TA-proteins selectively bound by HisTrx-SGTA. RAMP4 - stress-associated endoplasmic reticulum protein 1; Syb2 - synaptobrevin 2; Synt5 - syntaxin 5. B As for (A) but total translation products (input) were resolved by SDS-PAGE and results visualised by phosphorimaging. When total products are analysed high amounts of haemoglobin in the RRL distort the migration of Sec61β and its variant.


RNAs coding for C99 variants and containing a stop codon introduced either after the complete coding sequence or before the predicted TMD were used . E As for ( D ) but total translation products were subjected to immunoprecipation with either anti - C99 antibody ( for the full - length variants ) or with anti - β - amyloid antibody ( for products lacking the TMD ) . A filled square indicates inefficiently translated APP - C99 - TMD immunoprecipitated with anti - β - amyloid antibody .

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RNAs coding for C99 variants and containing a stop codon introduced either after the complete coding sequence or before the predicted TMD were used. E As for (D) but total translation products were subjected to immunoprecipation with either anti-C99 antibody (for the full-length variants) or with anti-β-amyloid antibody (for products lacking the TMD). A filled square indicates inefficiently translated APP-C99-TMD immunoprecipitated with anti-β-amyloid antibody.


A Full - length polypeptides were translated in the presence of 2 μM HisTrx or HisTrx - SGTA using RNAs lacking a stop codon and either stabilised at the ribosome with cycloheximide ( CHX ) or released with puromycin ( puro ) . Reactions were incubated with HisPur Cobalt resin , bound proteins eluted at high imidazole concentration , resolved by SDS - PAGE and visualised by phosphorimaging Filled circles indicate slower migrating species of radiolabelled products selectively enriched in CHX - treated samples .

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A Full-length polypeptides were translated in the presence of 2 μM HisTrx or HisTrx-SGTA using RNAs lacking a stop codon and either stabilised at the ribosome with cycloheximide (CHX) or released with puromycin (puro). Reactions were incubated with HisPur Cobalt resin, bound proteins eluted at high imidazole concentration, resolved by SDS-PAGE and visualised by phosphorimaging Filled circles indicate slower migrating species of radiolabelled products selectively enriched in CHX-treated samples.


total translation reactions were treated with RNaseA and immunoprecipitated with anti - C99

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total translation reactions were treated with RNaseA and immunoprecipitated with anti-C99 antibody