Fig. 4: TGFβ stabilizes ID1 protein through preventing its Nur77-mediated interaction with and ubiquitylation by Smurf2.

a SW620 cells were treated with TGFβ (10 ng/mL) for the indicated time. IB and qRT-PCR were applied to examine protein and ID1 mRNA expressions, respectively. hr hour. Two-tailed unpaired Student’s t test were used for statistical analysis, and data are presented as means ± SD (n = 3 biologically independent samples). b Cycloheximide (CHX) chase assay to determine ID1 turnover in the presence of TGFβ (10 ng/mL) in SW620 cells. min minute. c–e, g–i SW620 cells were transfected with the indicated expression plasmids, siRNAs, or shRNAs followed by MG132 treatment for 2 h and then TGFβ (10 ng/mL) treatment for 1 h. Protein ubiquitylation was examined by IP using the indicated antibodies and IB using anti-Ubiquitin (Ub) antibody. LC light chain, HC heavy chain, sh-ctr control shRNA, sh-Nur77 Nur77 shRNA. f SW620 cells were transfected with siRNAs before treatment with TGFβ (10 ng/mL) for the indicated times. Protein expressions were examined by IB. si-ctr control siRNA, si-Nur77 Nur77 siRNA. j SW620 cells transfected with the indicated shRNAs were treated with MG132 for 2 h and then TGFβ (10 ng/mL) for 1 h. Co-IP was applied to detect protein interactions. Data represent at least two independent experiments. Source data are provided as Source Data file.