Fig. 3: USP14 removed K48-linked polyubiquitin moieties from TAZ.
A, B The ubiquitination assay was performed with exogenously overexpressed TAZ (FLAG-tagged), Ub (MYC-tagged) and shUSP14/USP14-HA /USP14C114A (HA-tagged) in HEK293T cells. After 48 h, the cells were treated with MG132 (10 µM) for 6 h, cell lysates were collected, and immunoprecipitation was performed to analyse the ubiquitination of TAZ. C, D HEK293T cells were cotransfected with Ub-Myc and shUSP14, and treated with MG132 (10 µM) for 6 h after 48 h of transfection, and immunoprecipitation was performed to analyse the ubiquitination of TAZ. E In the in vitro ubiquitination assay, HEK293T cells were transfected with the Ub-Myc and TAZ-Flag plasmids. After 48 h, the cells were treated with MG132 (10 µM) for 6 h and purified with anti-Flag magnetic beads. Then, purified USP14-HA and USP14C114A-HA were incubated with purified TAZ protein in deubiquitination buffer for 2 h in a water bath at 37 °C. F PANC-1 cells with stable overexpression of TAZ-Flag were separated into the nucleus and cytoplasm by nuclear and cytoplasmic protein extraction kits, Co-IP experiments were conducted, and the stabilization of TAZ in the nucleus or cytoplasm by USP14 was detected. G, H The ubiquitination assay was performed with exogenously overexpressed TAZ (TAZ-FLAG), Ub (Ub-MYC/K63R-MYC/K48R-MYC/K63O-MYC/K48O-MYC) and USP14 /USP14C114A (HA-tagged) in HEK293T cells. Cells were treated with MG132 (10 µM) for 6 h after 48 h of transfection, and immunoprecipitation was performed to analyse the ubiquitination of TAZ.
