Figure 4.

Association of p55Cdc with Mad2 and Cdc27 during a normal cell cycle. Cells were arrested by a double-thymidine block and released as described in Materials and Methods. Samples were taken at the indicated time points (lanes 4–18), and protein extracts and nuclei (for FACS analysis) were prepared. (A) Coimmunoprecipitations. Extracts from cycling (lane 2) and nocodazole-arrested cells (lanes 1 and 3) were included as controls. Immunoprecipitations were done with either Mad2 antibody or Cdc27 antibody. The Mad2 immunoprecipitates were analyzed for coprecipitating p55Cdc (Top) and the Cdc27 immunoprecipitates were analyzed for coimmunoprecipitating p55Cdc (Middle) and Mad2 (Bottom) by Western analysis. The appropriate preimmune control assays were performed on extracts derived from nocodazole-arrested cells (lane 1, p.i.-noc). FACS analysis (B) and cdc2/cyclin B kinase assays (C) were performed at the indicated time points. (D) p55Cdc associated with Mad2 (lane 19) and Cdc27 (lane 20) as well as total p55Cdc in the cells (lane 21) 11 hr postrelease are shown. Five hundred micrograms protein extract was used for each immunoprecipitation.