Fig. 2.

USP28 interacts with MAST1 and extends its half-life. A The interaction between endogenous USP28 and MAST1 protein was analyzed in A549 by immunoprecipitation and immunoblotting using the specific antibodies. B Interaction between ectopically expressed USP28 and MAST1 was analyzed in HEK293 cells. Cells lysates were immunoprecipitated using Myc or Flag antibodies and analyzed by western blot. GAPDH was used as a loading control. C A549 cells were subjected to the Duolink PLA assay to analyze the interaction between USP28 and MAST1 using specific antibodies. The in situ USP28-MAST1 interaction (red PLA dots) was observed when USP28 and MAST1 were immunostained together but not when they were stained with individual antibodies. Scale bar: 10 µm. D–E The effect of USP28 and USP28CA on the half-life of Myc-MAST1 in HEK293 (D) and endogenous MAST1 (E) protein in A549 cells. CHX (150 μg/mL) was administered for the indicated time, and the cells were then harvested for western blotting with the indicated antibodies. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control. Data are presented as the mean and standard deviation of three independent experiments (n = 3). A two-way ANOVA followed by Tukey's post hoc test was used, and P values are indicated