Journal: Scientific Reports

Article Title: Functional kinomics establishes a critical node of volume-sensitive cation-Cl − cotransporter regulation in the mammalian brain

doi: 10.1038/srep35986

Figure Lengend Snippet: ( A ) Characterization of HEK293 cells with doxycycline (dox)-inducible MYC-KCC3 expression used in the RNAi screen. KCC3 wild type (WT) and KCC3 Thr 991 Ala/Thr 1048 Ala protein expression was induced by 0.1 μg/ml doxycycline in the culture medium for 24 hours . Cell lysates were subjected to Western immunoblot (IB) analysis with the indicated antibodies ( B ) Characterization of anti-KCC3 P-Thr 991 and anti-KCC3 P-Thr 1048 phospho-specific antibodies. 36 hours post-transfection with the indicated FLAG-tagged constructs, HEK293 cells were treated for 30 min with either isotonic conditions or hypotonic high K + conditions. Total cell extracts were subjected to IB analysis with the indicated antibodies. Mutation of these residues to alanine (Ala 991 and Ala 1048 ) prevented phosphorylation and eliminated the phospho-specific antibody signal at both sites. ( C ) Scheme of the RNAi screen using the human Dharmacon SMARTpool siRNA kinome library to identify essential kinase regulators of KCC3 Thr 991 phosphorylation. ( D ) Example of results from the primary siRNA screen. Band density of KCC3 P-Thr 991 from Western blots was quantitated by ImageJ software, and these values were used to calculate the magnitude of KCC3 P-Thr 991 increase or decrease by comparing to values derived from Firefly (FF) luciferase negative controls. The heat map depicts the average scores for each kinase siRNA pool in the screen that decreased (green) or increased (red) the signal of KCC3 P-Thr 991 relative to that of the FF siRNA. See Methods for further details. ( E ) Scattered and sorted robust z-scores of kinase hits from the siRNA primary screen. Several siRNA pools led to a significant decrease in the KCC3 P-Thr 991 signal (>50%, p < 0.01 compared to FF siRNA negative control). ( F ) Summary of kinase hits from the secondary siRNA screen. siRNAs targeting primary screen hits were analyzed for their ability to decrease KCC3 P-Thr 991 without affecting total KCC3 level. The (KCC3 P-Thr 991 )/(total MYC-KCC3) ratio was calculated for each target based on the quantification of immuno-reactive signals in triplicate Western blots, with a value of 100% for FF. Ratios were compared by one-way ANOVA (n = 3, mean ± SEM), with p < 0.01 considered statistically significant.

Article Snippet: To identify genes required for KCC3 P-Thr 991 phosphorylation, a high-throughput RNAi screen was performed in 24-well plates with the human Dharmacon SMARTpool siRNA kinome library targeting 541 kinases and kinase-related genes in which each mRNA is targeted by a pool of siRNAs consisting of a combination of four siRNA duplexes directed at different regions of the gene.

Techniques: Expressing, Western Blot, Transfection, Construct, Mutagenesis, Phospho-proteomics, Software, Derivative Assay, Luciferase, Negative Control