Figure 1.

BimEL is phosphorylated and targeted for degradation at mitosis. (a) Immnoblot analysis of HeLa cells synchronized using the thymidine/nocodozole (Thy/Noc) protocol as shown (top). Whole-cell extracts were analyzed for expression of BimEL following release from Thy/Noc for the indicated times. Immunoblots for three other proteins known to be degraded during mitosis, cyclin B1, Securin, and Polo-like kinase 1 (Plk-1) were performed for comparison. Anti-phospho histone H3 (P-H3) is included as a marker for onset of mitosis and APC2 as a loading control. Flow cytometry was used to confirm cell cycle phase (bottom). (b) Immunoblot analysis of HeLa cells synchronized with the cdk1 inhibitor RO3306 and released for the indicated length of time or left asynchronous (Asyn). Whole-cell extracts were analyzed by immunoblot analysis for expression of BimEL and three other proteins known to be degraded during mitosis: cyclin B1, cyclin A2, and cdc20. (c) Immunoblot of HeLa cells synchonized as in a and released for 90 min in the presence of DMSO (vehicle control) or MG132. (d) Immunoblot analysis of endogenous BimEL in whole-cell extracts treated with λ phosphatase (+) or control (−) prepared from nocodozole-treated HeLa cells