Fig. 4

OTUD3 inhibits TGF-β signaling and EMT by deubiquitinating SMAD7. A The effect of OTUD3 overexpression on CAGA-Luc transcriptional response in SMAD7-deficient HEK293T and control cells treated with TGF-β (2.5 ng/ml) for 16 h. B-C The effect of SMAD7 or OTUD3 overexpression on cell invasion in OTUD3-deficient (B) or SMAD7-deficient (C) MDA-MB-231 and control cells was detected by transwell assay. Left is a cell picture, enlarged images were taken using a 10× magnification lens; right is a statistical analysis. D Purified SMAD7 interacts with OTUD3-wt/CA in vitro. The left image shows the immunoprecipitation of purified SMAD7 protein with OTUD3 antibody, and the right image shows the immunoprecipitation of purified OTUD3 protein with SMAD7 antibody. The results were shown by immunoblotting. E HEK293T cells were transfected with Myc-OTUD3 and Flag-SMAD1-7 vectors, respectively, and TCL and immunoprecipitants were extracted for IB analysis. F To detect the correlation between OTUD3 and SMAD7, the anti-SMAD7 immunoprecipitate of HEK293T cells was subjected to IB, and 5% TCL was used as input. G-I OTUD3 deubiquitinats polyubiquitination of SMAD7 in vitro. G FLAG-SMAD7 and HA-Ub were transfected into HEK293T cells, respectively. The polyubiquitinized SMAD7 was immunoprecipitated with anti-FLAG M2 magnetic beads and incubated with purified OTUD3-wt/CA protein for different times. The cleavage products were analyzed by IB with antibodies HA-Ub, SMAD7 and OTUD3. H FLAG-SMAD7 and His-Ub were introduced into HEK293T cells, respectively. Subsequently, polyubiquitinated SMAD7 was extracted from the cell lysate, pulled off with nickel beads and incubated with purified OTUD3-wt/CA for 60 min. The cell lysate was analyzed by IB with SMAD7 and OTUD3 antibodies, respectively. The asterisk indicates that free SMAD7. I At 37℃ in vitro, His-SMAD7 was first incubated with E1,E2(UbcH6), GST-RNF12 and ubiquitin for 3 h, and then immunoprecipitated with SMAD7 antibody. Then it was incubated with purified OTUD3-wt /CA for 60 min, and the mixture was analyzed with Ub, SMAD7 and OTUD3 antibodies, respectively. (*p < 0.05;**p < 0.01;*** p < 0.001)