Effects of culture temperature on p16 protein expression, p16 binding to CDK4 and CDK6, CDK4 and CDK6 kinase activities, and phosphorylation of pRB in JKB p16MT cells. JKB p16MT cells, JKB p16 WT cells, and JKB p16 control cells were cultured under varying temperatures (40°C, 37°C, 34°C, and 31°C) for 1 day. (A) Total lysates of JKB p16MT cells were immunoprecipitated with anti-p16 MoAb and immunoblotted with the same MoAb. Total lysates of JKB p16MT cells (B) or JKB p16WT cells (C) were immunoprecipitated with anti-CDK2, anti-CDK4, or anti-CDK6 Abs and immunoblotted with anti-p16 MoAb. (D) Total lysates of JKB p16MT cells and JKB control cells were immunoprecipitated with anti-CDK4 or anti-CDK6 Abs. GST-RB fusion protein was used as substrate in assays of kinase activities of CDK4- or CDK6-associated immune complexes. (E) Total lysates of JKB p16MT cells and JKB control cells were immunoprecipitated with anti-pRB MoAb (Ab-1) and immunoblotted with anti-pRB MoAb (Ab-6).