Figure 3.

USP4 is a deubiquitinase of Twist1 protein. (A) H1975 cells stably expressing USP4 or a vector control were subjected to Q-PCR analyses. ns stands for no significance. (B) H1975 cells stably expressing shUSP4 or shGFP were treated with 50 μg/mL cycloheximide (CHX) for an indicated time interval, and then subjected to Western blot analyses. Twist1 protein levels were quantified and the plot was presented. (C) H1975 cells stably expressing shRNAs against USP4 (shUSP4) were treated with or without 20 μM MG132 for 12 h prior to Western blot analyses. (D) HEK293T cells were transfected with Flag-USP4 expressing plasmids for 36 h. Cells were treated with 20 μM MG132 for 6 h prior to Co-IP experiments. (E) HEK293T cells were co-transfected with Twist1 and His-ubiquitin in the presence of either Flag-USP4 or Flag-USP4^C311A expressing plasmids for 36 h (left panel). HEK293T cells were co-transfected with His-ubiquitin and either Flag-USP4 or Flag-USP4^C311A expressing plasmids for 36 h (right panel). Cells were then treated with 20 μM MG132 for 6 h prior to immunoprecipitation (IP) and Western blot analyses.