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

Journal: PLoS ONE

Article Title: Alba-Domain Proteins of Trypanosoma brucei Are Cytoplasmic RNA-Binding Proteins That Interact with the Translation Machinery

doi: 10.1371/journal.pone.0022463

Figure Lengend Snippet: (A) A panel of RNAi cell lines was constructed to knock down single Alba proteins or combinations of Alba proteins. A derivative of AnTat 90-13 , in which one copy of the GPEET coding region was replaced by enhanced GFP, was used as the parental line for Alba 1, Alba2, Alba1&2 and Alba3&4 RNAi cells. Unmodified AnTat 90-13 was used as the parental line for Alba 3 and Alba4 RNAi cells. RNAi was induced by addition of tetracycline to the cultures 3 days prior to the preparation of protein extracts. Band shift assays with 32 P-labeled GPEET were performed as described in the legend to . - Tet: uninduced; + Tet: induced; RNA only: incubation of GPEETLII without protein extract. (B) and (C): Alba1 and Alba2 proteins are dependent on Alba3. Western blot analysis of Alba proteins after knockdown of Alba3 (B) or Alba4 (C) by RNAi. Protein extracts of uninduced (- Tet) and induced (+Tet) cells were prepared every second or third day for 12 days and Alba proteins were detected with specific antibodies. HSP60 served as a loading control.

Article Snippet: To verify that the Alba proteins were indeed components of the band shifts we established a series of inducible RNAi cell lines in AnTat90-13 or a derivative in which one copy of the GPEET coding region is replaced by GFP (G. Schumann Burkard, manuscript in preparation).

Techniques: Construct, Electrophoretic Mobility Shift Assay, Labeling, Incubation, Western Blot

Journal: PLoS ONE

Article Title: Alba-Domain Proteins of Trypanosoma brucei Are Cytoplasmic RNA-Binding Proteins That Interact with the Translation Machinery

doi: 10.1371/journal.pone.0022463

Figure Lengend Snippet: (A) Total RNA from induced (+ Tet) and uninduced (- Tet) RNAi cells was extracted on the days indicated. Blots were hybridised with probes recognizing GPEET mRNA and 18S rRNA, which served as a loading control. (B) Quantification after normalisation to 18S rRNA. Steady-state levels of GPEET mRNA remained constant in the Alba4 and Alba3&4 RNAi cell lines.

Article Snippet: To verify that the Alba proteins were indeed components of the band shifts we established a series of inducible RNAi cell lines in AnTat90-13 or a derivative in which one copy of the GPEET coding region is replaced by GFP (G. Schumann Burkard, manuscript in preparation).

Techniques: