ZNF598 is destabilized in USP9X KO cells. (A) HCT116 or HCT116 USP9X^−/0 lysates were analyzed by immunoblot with the indicated antibodies (representative of three independent experiments). (B) Quantitative RT-PCR reactions for ZNF598, USP9X, and USP9Y (normalized to Actin) were performed with cDNA derived from the indicated cell lines. The mean of three independent biological replicates is shown, and error bars indicate the standard deviation. (C) HCT116 or HCT116 USP9X^−/0 cells were transfected with 0, 0.2, 0.4, 0.8, or 1.6 µg HA-ZNF598 and 0.2 µg GFP as a transfection control, and lysates analyzed by immunoblot with the indicated antibodies. Graph shows HA-ZNF598 relative to cotransfected GFP. Panel is representative of three experiments. (D) HCT116 or HCT116 USP9X^−/0 cells were transfected with 0.2 µg HA-ZNF598 and treated for the indicated times with 100 µg/ml cycloheximide (Chx). Lysates (8 µg for HCT116 and 20 µg HCT116-USP9X^−/0) were probed with the indicated antibodies. Graph represents the results from four independent experiments. Error bars represent the standard deviation. TPS, total protein stain.