Journal: Molecular and Cellular Biology
Article Title: Glycogen Synthase Kinase 3?-Mediated Serine Phosphorylation of the Human Glucocorticoid Receptor Redirects Gene Expression Profiles ▿
Figure Lengend Snippet: GSK-3β phosphorylates GR on Ser404. (A) Alignment of human GR sequence with the consensus site for GSK-3 kinase substrates. (B and C) U-2 OS cells stably expressing WT-GR or S404A-GR were pretreated with 0 to 5 μM concentrations of the GSK3α/β inhibitor BIO for 1 h before incubation with 100 nM Dex for 1 h. Afterward, cell extracts were analyzed by Western blot analysis to determine the amount of Ser211 and Ser404 phosphorylation of the GR. (D) U-2 OS cells stably expressing WT-GR or A549 cells that endogenously express GR were treated with 100 nM Dex for 1 h. Cell lysates were immunoprecipitated using anti-GR antibodies, and immunoblots were probed with anti-GSK-3α and anti-GSK-3β antibodies. (E) WT- and S404A-GR were immunoprecipitated from the stable U-2 OS cell lines. Immunoprecipitates were incubated with active recombinant GSK-3β, and GSK-3β-mediated phosphorylation of GR was visualized by autoradiography. (F) WT-GR-expressing U-2 OS cells transiently overexpressing WT, constitutively active (S9A), or kinase-dead (Y216F) versions of GSK-3β were lysed and assayed by Western blotting to determine pSer404-GR levels (*, P
Article Snippet: All proteins were electrophoretically immobilized to nitrocellulose membranes, which were subsequently probed with anti-GR antibodies (1:1,000; BD Biosciences), anti-GR 57 antibodies (1:500), anti-pSer404 GR antibodies (1:1,000), anti-pSer211 GR antibodies (1:2,000), anti-β-actin antibodies (1:2,500; Chemicon, Billerica, MA), anti-Bcl-xL antibodies (1:1,000; Chemicon), anti-GSK-3β antibodies (1:500; Cell Signaling, Danvers, MA), anti-GSK-3α antibodies (1:500; Chemicon), anti-p65 (RelA) antibodies (1:500; Cell Signaling), anti-CBP/p300 antibodies (1:500; Santa Cruz Biotechnologies, Santa Cruz, CA), anti-GRIP1 antibodies (1:500; Chemicon), anti-cIAP antibodies (1:1,000; Cell Signaling), anti-GAPDH antibodies (1:500; Cell Signaling), anti-nucleolin antibodies (1:1,000; Santa Cruz Biotechnologies), or anti-survivin antibodies (1:1,000; Stressgen, Ann Arbor, MI).
Techniques: Sequencing, Stable Transfection, Expressing, Incubation, Western Blot, Immunoprecipitation, Recombinant, Autoradiography