Figure 6.

 Cell cycle-regulated association of APC with hCDC20, hCDH1, and hMAD2. HeLa cells were synchronized at the prometaphase by a thymidine–nocodazole block. Cells were collected immediately after nocodazole treatment (lane 3, N), or released into fresh medium for 0.5, 1, 1.5, 4, and 10 hr (lanes 4–8). Asynchronous cells (lane 1, A) and cells arrested at G₁/S boundary by a double thymidine block (lane 2, T) were included as controls. (A) HeLa cells were lysed with the SDS sample buffer and the levels of cyclin B1, CDC27, hCDC20, hCDH1, and hMAD2 proteins were determined by Western blot analysis. The arrowhead in panel III points to hCDC20. The band above is a cross-reacting protein. (B) Extracts from HeLa cells were immunoprecipitated with either anti-hMAD2 antibody beads or with anti-CDC27 antibody beads, and the immunoprecipitates were analyzed by Western blotting with anti-APC2, anti-hCDC20, or anti-hCDH1 antibodies.