Figure 4

USP9X activity regulates Mcl-1 expression. (a) H1299 cells were treated with 10 μM WP1130 for 6 h and the subsequent downregulation of Mcl-1 was confirmed by western blotting. No change was observed in the Bcl-xL protein levels. The β-Actin expression levels were detected to normalize for protein loading. Proteins were quantified using the UN-SCAN-IT automatic digitizing system. Those intensities of the signals of Bcl-xl and Mcl-1 are relative to Actin. The mean ± SD represented the triplicate of three repeated western blotting. (b) A549 cells were plated in 10 cm tissue culture dishes and upon reaching 70% confluency were treated with 10 μg/ml CHX for 6 h or exposed to a combination of 7.5 μM WP1130 for 2, 4 and 6 h and 10 μg/ml CHX for 6 h. Mcl-1 and USP9X signals were then determined by western blotting. Proteins were quantified using UN-SCAN-IT. Those intensities of the signals of USP-9X and Mcl-1 are relative to Actin. (c) Mcl-1 proteins were immunoprecipitated from A549 cell lysates using anti-Mcl-1 antibody or control IgG. USP9X signals were then detected by western blotting. (d) A549 cells were exposed to a proteasomal inhibitor PS341 for 2, 4 and 6 h and the cells were then harvested and lysed. Mcl-1 was immunoprecipitated from these lysates with an anti-Mcl-1 antibody. USP9X signals and Mcl-1 signals were then detected by western blotting. Ub1 represents mono-ubiquitylated Mcl-1. Ub2 represents ubiquitylated Mcl-1 conjugated with two ubiquitins. Ub3 represents ubiquitylated Mcl-1 conjugated with three ubiquitins.