FIGURE 4:

TβRIII ectodomain shedding regulates the kinetics and magnitude of TGF-β signaling in MDA-MB-231 cells. (A) Lentiviral stable MDA-MB-231 cell lines expressing EV, WT-TβRIII, ΔShed-TβRIII, or Super-Shed TβRIII were plated in full serum medium and allowed to condition for 20 h before treatment with 50 pM of TGF-β1 for the indicated times. Western blot analysis was performed with indicated antibodies. Total Smad2 and β-actin were used as loading controls. Quantification of densitometric analysis is shown below as levels of phosphorylated Smad2/β-actin. Representative data from four independent experiments. (B) Summary of time-course experiment data. Densitometric analysis of phosphorylated Smad2/β-actin. Data for at least independent experiments for each time point shown as mean ± SEM. One-way ANOVA p < 0.05 for 15-m, 30-m, 1-h, 2-h, 3-h, 4-h, 5-h, and 6-h time points. (C) Integrated signaling over 6-h time course. Densitometric analysis of phosphorylated Smad2/β-actin of each experiment plotted as line graphs, with area under the curve calculated for each cell line. Data from four independent experiments shown as mean ± SEM. One-way ANOVA, p < 0.05. *Two-tailed t-test, p < 0.05.