Figure 7
GSK-3β regulation of p27 Kip 1 association with CDK2. A, SGC7901 cells were treated with (+) or without (-) 10 mM HMBA for 24 h. Protein extracts were immunoprecipitated with anti-CDK2 or anti-CDK4 antibodies. Normal rabbit IgG was used as the control. The CDK2-or CDK4-associated p27Kip1 in the resultant immune complexes was analyzed by western blotting using anti-p27Kip1 antibody with anti-CDK2 or -CDK4 antibody as loading controls. B&C, SGC7901 cells were pre-treated with (+) or without (-) 10 mM LiCl (B) or 10 μM SB-415286 (C) for 30 min followed by combination treatment with 10 mM HMBA for 24 h. Protein extracts were immunoprecipitated with an anti-CDK2 antibody. The CDK2 associated p27Kip1 in the resultant immune complexes was analyzed similar to A. D, SGC7901 cells were infected with Ad-HA-GSK-3βCA or vector control (Ad-β-gal) at an MOI of 10 pfu/cell. After 48 h incubation, whole cell protein was extracted and immunoprecipitated with anti-CDK2 antibody (upper panel). p27Kip1 was analyzed by western blotting similar to A. Overexpression of HA-tagged GSK-3CA was confirmed by western blotting using anti-GSK-3β antibody (lower panel). GSK-3β activity was assayed by an in vitro kinase assay using Snail protein as substrate (bottom panel).