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. 2009 Feb 17;29(8):2092–2104. doi: 10.1128/MCB.01405-08

FIG. 5.

FIG. 5.

GSK3β is required for phosphorylation of c-myc on T58, following its entry into the nucleus and collapse of AKT1 activity. (A) Untreated (−) E14 mESCs (plus LIF) were treated with LY294002 (40 μM) or LY294002 (40 μM) plus BIO (2 μM) for 24 h and were then subjected to immunostaining as indicated. Images were captured by confocal microscopy. Scale bar, 50 μm. (B) GSK3α−/− GSK3β−/− E14 mESCs were treated as for panel A with LY294002 and were probed with antibodies as indicated. (C) GSK3α−/− GSK3β−/− E14k mESCs (as for panel B) were transiently transfected with a GSK3βHA expression plasmid. Where indicated, cells were then treated with LY294002 (40 μM; 2 days posttransfection). Three days posttransfection, cells were probed with antibodies for HA and c-mycpT58, and staining was visualized by confocal microscopy. Scale bar, 50 μm. (D) WT E14k mESCs, E14k GSK3α−/− GSK3β−/− DKO mESCs, and DKO mESCs transfected with a GSK3βHA expression plasmid (as for panel C) were treated as indicated: untreated (−), treated with 40 μM LY294002 (+LY), or treated with 2 μM BIO (+BIO). Inhibitors were added 2 days posttransfection. Cell lysates were analyzed by immunoblot analysis following polyacrylamide gel electrophoresis and probed with antibodies as indicated.