10.1371/journal.pone.0119561.g006 Seung-il Choi Seung-il Choi Yong-Sun Maeng Yong-Sun Maeng Tae-im Kim Tae-im Kim Yangsin Lee Yangsin Lee Yong-Sun Kim Yong-Sun Kim Eung Kweon Kim Eung Kweon Kim Integrin-dependent endocytosis of TGFBIp in corneal fibroblasts. Public Library of Science 2015 rgd dystrophy type 2 gcd proteasome inhibitor MG 132 ecm TGFBIp trafficking 2015-04-08 04:16:31 Figure https://plos.figshare.com/articles/figure/_Integrin_dependent_endocytosis_of_TGFBIp_in_corneal_fibroblasts_/1372161 <p><b>A</b>. Endocytosis of TGFBIp was blocked by RGD peptide in a dose-dependent manner. Corneal fibroblasts were pre-incubated for 30 min in the absence (lane 1) or presence (lanes 2–4) of RGD or RAD peptides. TGFBIp (~1 μg/mL) was added to the medium and the cells were incubated for 120 min at 37°C. TGFBIp levels were measured by western blot analysis. <b>B</b>. TGFBIp interacts with integrin α<sub>V</sub>β<sub>3</sub> and α<sub>V</sub>. Cells were lysed with RIPA buffer and the lysate was immunoprecipitated with anti-integrin α<sub>V</sub>β<sub>3</sub> (left-hand panel) or anti-integrin α<sub>V</sub> (right-hand panel) antibody as indicated. Immunoprecipitates were resolved on 10% SDS-PAGE gels and immunoblotted with anti-TGFBIp polyclonal antibody. <b>C</b>. Co-localization of integrin α<sub>V</sub> with TGFBIp was visualized by confocal immunofluorescence microscopy. The merged images show TGFBIp as red, integrin α<sub>V</sub> as green, and areas of co-localization as yellow. The boxed area in the lower left-hand panel was magnified and is presented as the lower right-hand panel. Arrows identify regions of TGFBIp and integrin α<sub>V</sub> co-localization. Scale bars, 5 μm. <b>D</b>. Western blot analysis of HEK293T, NIH3T3, SK-N-MC, and ZW13-1 cell lines with monoclonal antibody against integrin α<sub>V</sub>. GAPDH was used as a loading control.</p>