FIG. 3.

Increasing association of p21 with cyclin A and cyclin B1 in G₂/M-phase cells. (A) Western blot analysis of cyclin complexes from lysates of synchronized Hs68 cells. Cyclin (Cyc) A and cyclin B1 complexes were immunoprecipitated (I.P.) from total lysates prepared from cells stimulated with serum for 15 h and cells released from aphidicolin block at the indicated time points. Immune complexes were separated on SDS–12% polyacrylamide gels, transferred to an Immobilon membrane, and detected by using the indicated antibodies (W.B. [Western blotting]) by ECL. Note that cyclin B1 immunoblots had to be exposed much longer than cyclin A immunoblots. (B) Comparative analysis of cyclin D1, cyclin A, and cyclin B1 immunocomplexes isolated from S-phase (pooled 0-, 3-, and 6-h time points)- and G₂/M-phase (pooled 10.5- and 13-h time points)-enriched cell lysates. The resulting immunocomplexes were resolved on the same SDS–12% polyacrylamide gel. Immunoblots were probed with either Cdk-specific antibodies (anti-Cdk4, anti-PSTAIRE for Cdk2 and Cdc2) or anti-p21, as indicated. Arrows with asterisks indicate differently phosphorylated species of p21.