( b ) Immunoprecipitation western blot for O - GlcNAc CK2α in HeLa after treatment with O - GlcNAcase inhibitor TMG for 12 h . Numbers indicate relative normalized for total CK2α concentrations .

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(b) Immunoprecipitation and western blot for O-GlcNAc CK2α in HeLa cells after treatment with the O-GlcNAcase inhibitor TMG for 12 h. Numbers indicate relative quantification normalized for total CK2α concentrations.


( c ) Western blot for total CK2α in HeLa following treatment with TMG for 12 h or 16 h . Numbers indicate relative normalized for loading control , mean values ( n = 4 ) shown ± s . d . Full gels corresponding to those in panels b c are in Supplementary Figures 14 15 .

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(c) Western blot for total CK2α protein in HeLa cells following treatment with TMG for 12 h or 16 h. Numbers indicate relative quantification normalized for loading control, mean values (n = 4) shown ± s.d. Full gels corresponding to those in panels b and c are in Supplementary Figures 14 and 15.


Phosphorimage of in vitro CK2 kinase assays with substrates . reactions were performed at 30 ° C . Numbers indicate ( nM ) of product for each kinase reaction . ( a ) AHCYL2 substrate ( 25 ng μl − 1 ) with 100 μM ATP 10 nM CK2α ± 11 nM CK2β for 6 min . ( b ) NAP1L3 substrate ( 15 ng μl − 1 ) with 100 μM ATP 10 nM CK2α ± 11 nM CK2β for 6 min . ( c ) NIPBL substrate ( 50 ng μl − 1 ) with 100 μM ATP 5 nM CK2α ± 11 nM Pin1 for 5 min . Full gels corresponding to those in panels a - c are in Supplementary Figures 19 25 .

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Phosphorimage analysis of in vitro CK2 kinase assays with protein substrates. All reactions were performed at 30 °C. Numbers indicate the concentration (nM) of product for each kinase reaction. (a) AHCYL2 substrate (25 ng μl−1) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min. (b) NAP1L3 substrate (15 ng μl−1) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min. (c) NIPBL substrate (50 ng μl−1) with 100 μM ATP and 5 nM CK2α ± 11 nM Pin1 for 5 min. Full gels corresponding to those in panels a-c are in Supplementary Figures 19 and 25.


( B ) of PIN2 phosphopeptide in our experiments . Bars represent mean plus standard error of replicates ( 2 biological replicates for nitrate treatments at 20 min ( Col - 0 roots ) 3 biological replicates for other experimental conditions ) . Each independent biological replicate consisted of a pool of 4 . 500 roots collected from Arabidopsis grown independently under same experimental conditions . asterisk indicates statistically significant differences in phosphoproteomic ( multiple t - comparison without testing corrections , assuming same variance , p < 0 . 05 ) .

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(B) Levels of PIN2 phosphopeptide in our experiments. Bars represent the mean plus standard error of replicates (2 biological replicates for nitrate treatments at 20 min (Col-0 roots) and 3 biological replicates for all other experimental conditions). Each independent biological replicate consisted of a pool of 4.500 roots collected from Arabidopsis plants grown independently under the same experimental conditions. The asterisk indicates statistically significant differences in phosphoproteomic analysis (multiple t-test comparison without testing corrections, assuming same variance, p < 0.05).


( C ) Detection of phosphorylation PIN2 by Phos - tag Western blotting . Arabidopsis ( Col - 0 ) were growth in ammonium as only nitrogen source , treated with 5 mM KNO3 or 5mM KCl as control . Total from roots were analyzed in SDS PAGE using Phos - tag to detect changes in phosphorylation status . Immunoblotting was performed with PIN2 antibody . Total isolated from eir1 . 1 roots were used as a negative control . red asterisks indicate a slow - or fast - mobility band corresponding to a more or less phosphorylated PIN2 , respectively .

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(C) Detection of phosphorylation PIN2 by Phos-tag Western blotting. Arabidopsis plants (Col-0) were growth in ammonium as only nitrogen source, and treated with 5 mM KNO3 or 5mM KCl as control. Total protein from roots were analyzed in SDS PAGE using Phos-tag to detect changes in phosphorylation status. Immunoblotting was performed with PIN2 antibody. Total proteins isolated from eir1.1 roots were used as a negative control. White and red asterisks indicate a slow- or fast-mobility band corresponding to a more or less phosphorylated PIN2, respectively.


( D ) Western blot against PIN2 comparing nitrate treated ( KNO3 ) control ( KCl ) condition in Arabidopsis roots for genotypes ( eir1 - 1 mutant background was complemented with PIN2 : : PIN2wt - GFP ( PIN2wt ) , PIN2 : : PIN2S439D - GFP ( PIN2S439D phospho - mimic point mutation ) or PIN2 : : PIN2S439A - GFP ( PIN2S439A , phospho - null point mutation ) .

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(D) Western blot against PIN2 protein comparing nitrate treated (KNO3) and control (KCl) condition in Arabidopsis roots for all genotypes (eir1-1 mutant background was complemented with PIN2::PIN2wt-GFP (PIN2wt), PIN2::PIN2S439D-GFP (PIN2S439D phospho-mimic point mutation) or PIN2::PIN2S439A-GFP (PIN2S439A, phospho-null point mutation).


( A ) root of Col - 0 wild type or eir1 - 1 mutant was measured using ImageJ program 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 10 - 15 roots each replicate ) . box plot shows data within interquartile range ( 25th 75th percentiles ) a solid black represents median . Whiskers show maximum minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Asterisk indicates statistically significant difference between analyzed by unpaired , two - assuming equal variance t - ( * * p < 0 . 01 ) .

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(A) Primary root length of Col-0 wild type plants or eir1-1 mutant plants was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 10-15 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicates statistically significant difference between analyzed by unpaired, two-tailed and assuming equal variance t-test (** p < 0.01).


( B ) Number of initiating emerging lateral roots of Col - 0 or eir1 - 1 mutant were counted using microscopy 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 10 - 15 roots each replicate ) . box plot shows data within interquartile range ( 25th 75th percentiles ) a solid black represents median . Whiskers show maximum minimum values no further than 1 . 5x IQR ( interquartile range ) . Asterisk indicates statistically significant difference between analyzed by unpaired , two - assuming equal variance t - ( * * p < 0 . 01 ) .

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(B) Number of initiating and emerging lateral roots of Col-0 or eir1-1 mutant plants were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 10-15 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Asterisk indicates statistically significant difference between analyzed by unpaired, two-tailed and assuming equal variance t-test (** p < 0.01).


( A ) root of different genotypes was measured using ImageJ program 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 8 - 10 roots each replicate ) . box plot shows data within interquartile range ( 25th 75th percentiles ) a solid black represents median . Whiskers show maximum minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Asterisk indicate statistically significant difference between means analyzed by unpaired , two - assuming equal variance t - ( * p < 0 . 05 , * * p < 0 . 01 ) .

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(A) Primary root length of the different genotypes was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 8-10 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicate statistically significant difference between means analyzed by unpaired, two-tailed and assuming equal variance t-test (*p < 0.05, **p < 0.01).


( B ) Number of initiating emerging lateral roots for genotypes were counted using microscopy 3 days after 5 mM KNO3 or KCl treatments . Tukey box plot show results from 3 independent biological replicates per experimental condition ( n = 8 - 10 roots each replicate ) . box plot shows data within interquartile range ( 25th 75th percentiles ) a solid black represents median . Whiskers show maximum minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Asterisk indicate statistically significant difference between means analyzed by unpaired , two - assuming equal variance t - ( * p < 0 . 05 , * * p < 0 . 01 ) .

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(B) Number of initiating and emerging lateral roots for all genotypes were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 8-10 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicate statistically significant difference between means analyzed by unpaired, two-tailed and assuming equal variance t-test (*p < 0.05, **p < 0.01).


( A ) Confocal microscopy using Zeiss Airyscan microscope Zeiss - blue3 . 1 software was used to visualize PIN2 in Arabidopsis roots . " e " denotes epidermis " c " cortex . Scale bar = 20 µm . ( B - D ) Tuckey box plots show PIN2 - GFP ( number of roots analyzed per experimental conditions = 8 - 10 ; arbitrary units , a . u . ) at total in roots ( B ) , from epidermal ( C ) cortex ( D ) plasma in different genotypes . box plot shows data within interquartile range ( 25th 75th percentiles ) a solid black represents median . Whiskers show maximum minimum values no further than 1 . 5x IQR ( interquartile range ) . Outlier data are plotted individually . Letters denote statistically significant difference between means as determined by one - way ANOVA followed by Tukey HSD post hoc ( p < 0 . 05 ) .

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(A) Confocal microscopy using Zeiss Airyscan microscope and Zeiss-blue3.1 software was used to visualize PIN2 in Arabidopsis roots. "e" denotes epidermis and "c" cortex. Scale bar = 20 µm. (B-D) Tuckey box plots show PIN2-GFP fluorescence intensity quantification (number of roots analyzed per experimental conditions = 8-10; arbitrary units, a.u.) at the total cell membrane in roots (B), and from epidermal (C) and cortex (D) plasma membrane in the different genotypes. The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Letters denote statistically significant difference between means as determined by one-way ANOVA analysis followed by Tukey HSD post hoc test (p < 0.05).