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. 2013 Aug 2;20(10):1393–1403. doi: 10.1038/cdd.2013.93

Figure 6.

Figure 6

Phosphorylation of the BimEL degron is mediated by Aurora A kinase during mitosis. (a) 293T cells were synchronized in mitosis as in Figure 1a and released into vehicle control (DMSO), MG132, RO3306, or the indicated concentration of MLN8054. Whole-cell extracts were analyzed by immunoblot to detect endogenous BimEL and phosphorylated BimEL on serines 93/94/98 (P-S93/94/98). Cdc27 immunoblots were used to monitor mitotic state of cells and β-actin as loading control. (b) HeLa cells were synchronized in G2/M with RO3306 and released into fresh media plus vechicle (DMSO), MG132 (10 μM), or MLN8054 (5 μM) and harvested 90 min post release. Whole-cell extracts were analyzed by immunoblot to detect endogenous BimEL. P-T210 Plk1 and total Plk1 were used as controls to confirm the downstream effects of the aurora A inhibitor MLN8054. (c) Knockdown of Aurora A expression in HeLa cells using two different siRNA. A non-silencing siRNA (Ctl) was used as control. Immunoblot analysis was performed against endogenous BimEL and Aurora A. P-T210 Plk1 and total Plk1 were used as a control to confirm the downstream effects of the knockdown. Cdc27 immunoblots were used to confirm mitotic state of cells and β-actin as loading control. (d) 293T cells were transfected with HA-tagged βTrCP1 or empty vector control (EV). Cells were synchronized in mitosis as in Figure 1a and released into vehicle control (DMSO), MG132 (10 μM), RO3306 (9 μM), MLN3306 (5 μM), or left untreated (UT). Immunoprecipitation (IP) was performed using anti-HA antibody. Immunoblot analysis was perfored on IPs to detect endogenous BimEL and transfected βTrCP1 (HA). Cell extracts used as input for IP were analyzed by immunoblot to measure expression of total BimEL, HA (βTrCP1), and β-Actin as loading control. (e and f) Recombinant GST-BimEL or phosphorylation site mutants produced in E. coli were incubated with increasing amounts of purified Aurora A kinase (e) or 500 ng purified Aurora A kinase (f). Reactions were analyzed by immunoblot to detect GST-Bim, P-S93/94/98 GST-BimEL, and Aurora A. (g) Recombinant GST or GST-BimEL produced in E. coli were incubated with either mitotic Hela or 293T extracts prepared using the standard thymidine/nocodazole protocol. Immunoblot analysis was performed to detect co-precipetated endogenous Aurora A. Mitotic Hela or 293T cell extracts were used as an input to measure expression of endogenous Aurora A. Image of Red Ponceau-stained membrane used to show GST proteins