Fig. 2: USP14 interacted with and stabilized TAZ in a deubiquitinase activity-dependent manner.
A Reciprocal Co-IP experiments were performed with the USP14/TAZ antibody in PANC-1 cells. The interaction of endogenous USP14 and endogenous TAZ was analysed by immunoblotting. B GST pull-down assay was performed to detect the interaction of USP14 and TAZ. C, D Plasmids containing full-length and truncated sequences of TAZ and USP14 were constructed, and HEK293T cells were transfected with the indicated plasmids. A Co-IP assay was performed to explore the binding regions between USP14 and TAZ. E SW-1990 cells were transfected with the USP14-HA and TAZ-Flag plasmids. After 48 h transfection, an immunofluorescence assay was conducted to acquire the images by using fluorescence microscopy. F PANC-1 cells were transfected with the USP14C114A-HA plasmid as indicated. Immunoblotting analysis was carried out to examine the protein expression of TAZ and USP14. G HEK293T cells were transfected with shScramble or shRNA targeting USP14. After 48 h, these cells were treated with or without MG132 (10 µM) for 6 h. Immunoblotting analysis was used to examine USP14 and TAZ expression. H, I PANC-1 cells transfected with the indicated plasmid were treated with CHX (50 μM) for the indicated time intervals. Cells were harvested, and the expression of TAZ and USP14 was measured by immunoblotting. The level of protein expression was quantified by densitometry and plotted. GAPDH was used as the internal reference, and the protein expression of TAZ was normalized to that at the t = 0 time point. The data were analysed by one-way ANOVA and were presented as the mean ± SD. See also Fig. S4.
