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, 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:

Journal: The EMBO Journal

Article Title: Lats2 kinase potentiates Snail1 activity by promoting nuclear retention upon phosphorylation

doi: 10.1038/emboj.2011.357

Figure Lengend Snippet: A screen for post-translational modifiers of Snail1 protein stability. (A) Stick figure representation of human Snail1-Clic Beetle Green (CBG) bioluminescent plasmid. NES: nuclear export signal region. (B) Bioluminescence and western blot of Snail1–CBG expressing HEK293 (clone #8) or untransfected control HEK293 cells, for the indicated proteins (right). (C) Bioluminescence of HEK293.Sn-CBG clone 8 transfected with Snail1 RNAi, GSK3β RNAi, or treated with the proteosome inhibitor MG132. Results are presented as bioluminescence relative to control untreated cells (set at an arbitrary value of 1.0). *Identifies the difference in Snail1 stabilizing effect between inhibition of GSK3β and inhibition of proteasome function. (D) Western blot analysis for the EMT marker E-cadherin (i.e., a Snail1 target gene) in HEK293.Sn-CBG clone 8 cells following transfection with control Luc RNAi, Snail1 RNAi, or GSK3β RNAi. (E) A human kinome RNAi screen (Qiagen) for proteins that stabilize or destabilize Snail1 protein level, as described in Materials and methods. Individual RNAi values are presented as Median Average Deviation (MAD) bioluminescence from the median of the complete library and Luc control RNAi. Control for maximum stabilization is MG1323 treatment (blue diamonds); for maximum destabilization is Snail RNAi (red circles); and for RNAi control is Luciferase RNAi (black triangles). The RNAi library results are in triplicate for each RNAi (orange diamonds). There were two RNAi's per kinase in the library. The blue broken lines identify ±3 MAD.

Article Snippet: * Identifies the difference in Snail1 stabilizing effect between inhibition of GSK3β and inhibition of proteasome function. ( D ) Western blot analysis for the EMT marker E-cadherin (i.e., a Snail1 target gene) in HEK293.Sn-CBG clone 8 cells following transfection with control Luc RNAi, Snail1 RNAi, or GSK3β RNAi. ( E ) A human kinome RNAi screen (Qiagen) for proteins that stabilize or destabilize Snail1 protein level, as described in Materials and methods.

Techniques: Plasmid Preparation, Western Blot, Expressing, Control, Transfection, Inhibition, Marker, Luciferase

Journal: The EMBO Journal

Article Title: Lats2 kinase potentiates Snail1 activity by promoting nuclear retention upon phosphorylation

doi: 10.1038/emboj.2011.357

Figure Lengend Snippet: Metastatic breast cancer cells contain increased amounts of Lats2 and in these cells Lats2 affects their invasive capacity. (A) Western blot analysis with the indicated antibodies of cell lysates from the non-transformed and non-tumourigenic human breast epithelial cell line MCF10A and two tumourigenic and metastatic human breast cancer cell lines MDA-MB-231 and BT549. (B) A Snail1 antibody western blot analysis of MDA-MB-231 cells transduced with lentiviruses expressing both a Snail1 shRNAi and various YFP-tagged, RNAi-resistant Snail1 mutants, as indicated. TA: T203A mutation; TE: T203E mutation; N or NLS: nuclear localization signal. (C, D) MDA-MB-231 cells as described in (B) were aggregated and placed in a 3D collagen I gel (2 mg/ml). Phase image from a representative imbedded cell aggregate shows the distance of invasion/migration of cells at 48 h. Magnified boxes show presence of cells leaving the aggregate. Results from multiple aggregates (@10) per well from multiple experiments (3) are quantified in (E) and depict the distance cells migrated centrifugally from the aggregates, relative to control cells arbitrarily set at 100%. Data are represented as mean±s.d. **Indicates P<0.01.

Article Snippet: * Identifies the difference in Snail1 stabilizing effect between inhibition of GSK3β and inhibition of proteasome function. ( D ) Western blot analysis for the EMT marker E-cadherin (i.e., a Snail1 target gene) in HEK293.Sn-CBG clone 8 cells following transfection with control Luc RNAi, Snail1 RNAi, or GSK3β RNAi. ( E ) A human kinome RNAi screen (Qiagen) for proteins that stabilize or destabilize Snail1 protein level, as described in Materials and methods.

Techniques: Western Blot, Transformation Assay, Transduction, Expressing, Mutagenesis, Migration, Control