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. 2016 Oct 12;7(48):78958–78970. doi: 10.18632/oncotarget.12586

Figure 1. Validation of phospho-specific Mcl-1 antibody, kinetics of Mcl-1 phosphorylation in response to vinblastine, and effect of MG132.

Figure 1

A. HeLa cells were untreated or treated with vinblastine (VBL) as indicated and whole cell extracts (WCE, lanes 1 and 2) or immunoprecipitated (IP) Mcl-1 (lanes 3 and 4) subjected to immunoblotting for Mcl-1 (upper panel) or with a phospho-specific antibody (p-Ab) originally developed to detect phospho-Bcl-xL (middle panel) or with a phosphorylation-independent Bcl-xL antibody (lower panel). B. HeLa cells were synchronized at the G1/S boundary by double thymidine block and were either untreated or treated with 30 nM vinblastine (VBL) 1 h after release, then harvested at the indicated times. In the untreated group, time points corresponding to late G2-M and G1 phases are indicated. Extracts were subjected to immunoblotting for the proteins indicated, with GAPDH used as a loading control. Phosphorylated Mcl-1 (P-Mcl-1) was detected with an antibody developed to detect phosphorylated Bcl-xL as in Figure 1A. C. HeLa cells were treated with 30 nM vinblastine (VBL) for 24 h, in the presence or absence of 20 μM MG132 added 4 h prior to harvest, and extracts prepared and subjected to immunoblotting for Mcl-1. In the left panel (Mcl-1 Ab-1), Mcl-1 antibody sc-12756 was used, and in the right panel (Mcl-1 Ab-2), Mcl-1 antibody sc-20679 was used.