Fig. 2: Smurf2 degrades SMAD2 through ubiquitination and then downregulates its expression.
From: Smurf2-induced degradation of SMAD2 causes inhibition of hair follicle stem cell differentiation
Smurf2 mRNA (A) and protein (B) levels in HFSCs after 7-day differentiation determined by RT-qPCR and western blot assay, with β-actin as internal reference, *p < 0.05 vs. day 0. C Interaction between Smurf2 and SMAD2 in HFSCs identified by IP. D Stability of SMAD2 protein in response to Smurf2 overexpression in HEK293 cells treated with cycloheximide for 1, 2, 4, 6, 8 h. *p < 0.05 vs. oe-SMAD2 + oe-NC group. E SMAD2 protein level in HFSCs overexpressing Smurf2 measured by western blot assay, with β-actin as internal reference, *p < 0.05 vs. oe-NC group. F Ubiquitination of SMAD2 in HFSCs after Smurf2 overexpression determined by IP (IB-Ub refers to ubiquitinated antibody used in immunoblot, while IB-SMAD2 refers to SMAD2 antibody used in immunoblot). G The ubiquitination of SMAD2 in HFSCs treated with proteasome inhibitor MG132 or DMSO alone or in the presence of Smurf2 determined by IP. *p < 0.05 vs. oe-NC + DMSO group; #p < 0.05 vs. oe-Smurf2 + DMSO group. H The ubiquitination of SMAD2 in HFSCs in response to MG132 treatment and SMAD2 overexpression or in the presence of Smurf2 determined by IP. *p < 0.05 vs. flag-SMAD2 + oe-NC + DMSO group; #p < 0.05 vs. flag-SMAD2 + oe-Smurf2 + DMSO group. Data are expressed as mean ± standard deviation. Data comparison between two groups was performed by unpaired t test, while data between multiple groups were compared by one-way ANOVA with Tukey’s post hoc test. Data at different time points were compared by two-way ANOVA with Bonferroni post hoc test. Each cell experiment was repeated 3 times.
