Fig. 5
TRIM25 mediates the ubiquitinated degradation of PPM1B. (A) WB analysis of PPM1B protein levels in AGS and MGC-803 cells transiently transfected with Flag-TRIM25 plasmid or mock control plasmid. (B) AGS and MGC-803 cells were transient transfected and cultured with Flag-TRIM25 plasmid or simulated control plasmid for 24 h, and then treated with CHX for 3, 6, 9 h, respectively. WB was used to detect the protein level of PPM1B in the transfected cells. (C) AGS cells were transfected with Flag-TRIM25 plasmid or mock control plasmid and cultured for 24 h, then further incubated with MG132 (10 µM) for 4 h or with chloroquine (25 µM) for 6 h, WB analysis of PPM1B protein levels in transfected cells. (D) Ubiquitination of PPM1B in HEK293T cells co-transfected with Flag-TRIM25, HA-UB, and Myc- PPM1B plasmids by Co-IP analysis. (E) Co-ip analysis of ubiquitination of PPM1B in HEK293T cells co-transfected with Flag-TRIM25 plasmid, Myc-PPM1B plasmid and HA-UB-K48 or HA-UB-K63 plasmid. (F) Co-IP analysis of PPM1B ubiquitination in AGS cells co-transfected with Flag-TRIM25 plasmid or Flag-TRIM25 truncated mutant (RING domain) plasmid, Myc-PPM1B plasmid and HA- UB plasmid.
