Journal: PLoS ONE
Article Title: GSK3β-Dependent Phosphorylation Alters DNA Binding, Transactivity and Half-Life of the Transcription Factor USF2
Figure Lengend Snippet: GSK3β-mediated phosphorylation of USF2 affects its transactivity, DNA binding and target gene expression. (A) HeLa cells were cotransfected with pFR-5Gal4-RE-Luc, an expression vector for constitutively active GSK3β-S9A and WT or mutant pcDNA6-Gal4-USF2 (1-231) or the appropriate empty Gal4 vector. The luciferase activity was calculated as fold induction compared to the Gal4-USF2 (1-231)-WT luciferase activity after subtracting the values from the empty Gal4 expression vector. *, significant differences control vs. GSK3β. (B) Representative Western blot of the transfected constructs. 50 µg of protein from transfected cells were probed with an antibody against Gal4, HA-tag and α-tubulin. (C) Quantitative RT-PCR analyses of FAS, HO-1, PAI-1 and USF2 mRNA levels in GSK3β +/+ and GSK3β −/− cells. *, significant differences WT vs. GSK3β −/− . (D) Western Blot analyses of FAS, HO-1 and PAI-1 expression in GSK3β +/+ and GSK3β −/− cells . 50 µg of protein were subjected to Western analysis with antibodies against FAS, HO-1, PAI-1 or β-catenin, c-Myc or α-tubulin; the latter served as a loading control. (E) ChIP was performed in GSK3β +/+ and GSK3β −/− cells with either USF2 antibody, control IgG or RNA Pol II antibody. The quantitative PCR was performed with primers amplifying the FAS, HO-1, and PAI-1 promoter containing the USF2 binding sites, and with primers amplifying the β-actin promoter binding RNA Pol II as outlined in Materials and Methods . *, significant differences WT vs. GSK3β −/− .
Article Snippet: In brief, after cross-linking, cell lysis and sonication, chromatin samples were incubated with anti-USF2 antibody (N-18, Cat.Nr. sc-861, Santa Cruz Biotechnology), anti-RNA polymerase II antibody (N-20, Cat.Nr. sc-899X, Santa Cruz Biotechnology) or IgG preimmune serum (BioScience) in an ultrasonicator bath for 1 h at 4°C; after centrifugation, the precleared samples were mixed with protein G Sepharose beads for 1 h at 4°C.
Techniques: Binding Assay, Expressing, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Western Blot, Transfection, Construct, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction