Figure 3.

BimEL is ubiquitinated at mitosis and requires phosphorylation on Serine 93/94/98. (a) 293T cells were co-transfected with HA-tagged Ubiquitin (Ub) and Flag-tagged BimEL, BimL BimS, or empty vector control (EV). Cells were synchronized as shown in Figure 1a. Asynchrous (Asyn) cells treated with PMA and MG132 was used as a positive control for BimEL ubiqutination (lanes 1 and 9). Cells were treated with MG132 (+) or vehicle control (−) as indicated. (Left) Anti-Flag immunoprecipitation (IP) followed by immunoblot for HA and FLAG epitopes. (Right) Immunoblots were performed on cell extracts used as input for IPs using anti-Flag and HA antibodies to detect Bim and Ub expression. Anti-Cdc27 immunoblot was used to confirm mitotic state of cells and β-actin was monitored as loading control. (b) Nocodozole-treated 293T cells were co-transfected with HA-Ub and Flag-tagged wild-type (WT) BimEL, S69A, S94/98A, or EV control. Anti-Flag IP was performed as in a. Resulting IPs were analyzed by immunoblot using anti-Flag, anti-HA, and the phoshospecific BimEL antibodies (P-S93/94/98 and P-S69). Cells were treated with MG132 (+) or vehicle control (−) as indicated. Immunoblots were performed on cell extracts used as input for IPs using anti-Flag to confirm expression of BimEL constructs and β-actin was monitored as loading control. (c) 293T cells were transfected with WT BimEL or phosphorylation site mutants as indicated. In all cases, EGFP was cotransfected to monitor transfection effeciency. Cell extracts were prepared from either asynchronous (A) or after nocodozole (N; 100 ng/ml) treatment. Immunblots were performed on WCE using anti-Flag to detect BimEL, β-actin as loading control, EGFP to monitor transfection efficiency, and cdc27 to confirm the mitotic state of the cell