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. 2010 Jun;24(6):1136–1150. doi: 10.1210/me.2009-0466

Fig. 3.

Fig. 3.

GSK3α interacts with GR. A, The effect of Dex treatment on GSK3 phosphorylation. PD1.6 cells were untreated or treated with 100 nm Dex for the indicated time intervals and processed for Western blotting (WB) using antibodies to phospho-Ser9 GSK3β, phospho-Tyr216 GSK3β, GSK3β, or α-Tubulin. B, GSK3α interacts with native GR and is released upon Dex treatment. Thymocytes were untreated or treated with 100 nm Dex for the indicated time intervals. Total lysates were immunoprecipitated (IP) using M20 antibodies specific to GR (Santa Cruz Biotechnology) and Protein A beads. The one-step Genscript IP-WB kit was used for Western blot analysis to prevent the detection of heavy chain. Nevertheless, a band of the heavy chain is seen, as indicated. Antibodies to GSK3α/β (0011-Α, Santa Cruz Biotechnology) were applied. For the IP analysis, two pre-IP samples (lanes 1 and 9; input 1:40 of IP samples) were run to display the location of GSK3α and -β. Lane 2 contains only the antibody (Ab), which provides the location of the heavy chain. Samples of total lysates were taken before immunoprecipitation (Pre-IP), to show the initial expression levels of the indicated proteins. C, GR interacts with GSK3α in PD1.6 T lymphoma cells. PD1.6 cells were untreated (lane 1), or treated with SB216763 and/or Dex for 1 h. Immunoprecipitation was performed as described in panel B. Panels a and b are two different exposures. The GSK3α band appears just above the heavy chain band. D, Selective binding of GSK3α to GR. MCF-7 cells were transfected with 5 μg of plasmids encoding hGRα (lanes 1–3), hGSK3α (lanes 2 and 4), and/or hGSK3β.HA (hemagglutinin-labeled) (lanes 3 and 5). Empty pcDNA3 vector (5 μg) was included in samples 1, 4, and 5 to achieve equal amounts of transfected DNA. Cell lysates were immunoprecipitated with M20 antibodies against GR, and the presence of coimmunoprecipitated GSK3 was analyzed by WB as described in panel B. E, Long-term inhibition of GSK3 leads to reduced GR expression. PD1.6 cells were treated with SB216763 for 2 and 24 h, and the GR expression level was analyzed on Western blot. β-Catenin expression is shown as indication for GSK3 inhibition.