Figure 4
TGF-β1 induced Smad3 docking to membrane for activation.
(a,b) Dual-view TIRFM images of p-Smad3 (pAb, Alexa-488) and Myc-TRI (Myc mAb, Alexa-555) before (a) and after (b) TGF-β1 stimulation in the Myc-TRI expressing HeLa cells. The cells were immunostained with antibodies against Myc and p-Smad3. Scale bars: 5 μm. (c) Quantification of colocalization of p-Smad3 with Myc-TRI using the Mander’s coefficient (Blobprob plugin, ImageJ). 0.98% ± 0.07% (n = 6 cells; mean ± SEM) and 11.98% ± 0.67% (n = 6 cells; mean ± SEM) of p-Smad3 molecules were colocalized with Myc-TRI in the absence and presence of TGF-β1, respectively. (**p < 0.01, t-test). (d) Western blotting analyses of p-Smad3, Smad3 and TRI. HeLa cells were treated with 10 ng/ml TGF-β1, harvested at the indicated time points (0, 1, 5, 10, 30, 60, 120 min), and then subjected for western blotting analyses of p-Smad3, Smad3 and TRI in the membrane extraction. Na/K ATPase was used as the internal control for membrane extraction. (e) Co-IP analysis of the interaction between TRI and Smad3 or p-Smad3 using the membrane fractions from HeLa cells. The cells were treated with 10 ng/ml TGF-β1, harvested at the indicated time points (0, 1, 5, 10, 30, 60, 120 min), and subjected for co-IP analysis.
