Fig. 3

OTUD3 maintains SMAD7 stability through deubiquitylation. A HEK293T cells were transfected with HA-Ub, Flag-SMAD7, Myc-OTUD3-wt/CA, and treated with MG132 (5 µM) for 4 h, while the control group was treated with DMSO. The total cell lysate (TCL) and immunoprecipitates were extracted for IB. HA-Ub labeled HEK293T cells were transfected with Flag-SMAD7, Myc-RNF12, and Myc-OTUD3-wt/CA (B) or OTUD3 shRNA (#1 and #2) (C), respectively. IB of TCL and immunoprecipitates were peformed. D Transfecting Myc-OTUD3 plasmid into HA-Ub labeled OTUD3-/- and OTUD3+/+ HEK293T cells respectively, then TCL and immunoprecipitates of transfected and control cells were extracted for IB, and endogenous polyubiquitination SMAD7 was immunoprecipitated with anti-SMAD7 antibody. E Cells expressing empty vector (Co.vec), Myc-OTUD3-wt or Myc-OTUD3-CA were treated by cycloheximide (CHX, 20 µg/ml) for different time, and lysates were collected for IB to detect the turnover rate of SMAD7 protein