Ectopic expression of p27^KIP1 stabilizes v-cyclin and CDK6. (A) U2OS cells were cotransfected with expression vectors for Myc-v-cyclin, HA-CDK6, and different amounts of p27^KIP1 (0.7, 0.1, 0.05, and 0.025 μg). Cell lysates were immunoprecipitated with anti-Myc antibody and associated proteins were analyzed on SDS-PAGE (12%) by immunoblotting with anti-Myc, anti-CDK6, and anti-p27^KIP1 antibodies (3 top panels). Total lysates of transfected cells were resolved by SDS-PAGE and immunoblotted with anti-Myc, anti-CDK6, anti-p27^KIP1, and antiactin antibodies (4 lower panels). (B) Coexpression of p27^KIP1 protects cells from v-cyclin-CDK6-induced cell death. U2OS cells were cotransfected with expression vectors for Myc-v-cyclin alone or together with HA-CDK6 and p27^KIP1 (0.7 μg). Cells were analyzed 48 hours after transfection by indirect immunofluorescence. The cells were labeled by anti-Myc and anti-CDK6 antibodies and their nuclear morphology was visualized by Hoechst staining. Cells showing healthy Myc-v-cyclin and CDK6 (endogenous or HA-tagged)-expressing cells are indicated by arrows, whereas arrowheads are pointing at apoptotic cells. (C) U2OS cells were cotransfected with Myc-v-cyclin, HA-CDK6, and p27^KIP1 or p21^CIP1 (left) or with cyclin D2, HA-CDK6, and p27^KIP1 or p21^CIP1 (right). Cell lysates were immunoprecipitated with anti-Myc or anti-cyclin D2 antibodies and associated proteins were analyzed on SDS-PAGE (12%) by immunoblotting with anti-CDK6, anti-p27^KIP1, and anti-p21^CIP1 antibodies. — indicates no inhibitors (A,C).