Fig. 6. Association of the nectin-afadin complex with PDGF receptor. (A) Colocalization of the nectin-afadin complex with PDGF receptor at cell-cell adhesion sites. Confluent NIH3T3 cells (Aa) or nectin-3-knockdown NIH3T3 cells (Ab) were immunostained with the anti-PDGF receptor pAb (Santa Cruz Biotechnology) and the rat anti-nectin-3 or mouse anti-afadin mAb. Arrowheads indicate the colocalization sites of nectin-3 or afadin with PDGF receptor. Scale bars: 10 µm. (B) Recruitment of nectin-3 and PDGF receptor at the contact sites between Nef-1-coated beads and NIH3T3 cells. NIH3T3 cells were incubated with Nef-1-coated microbeads (Ba) or ConA-coated microbeads (Bb) for 1 hour; they were then fixed and immunostained with the anti-PDGF receptor pAb and the anti-nectin-3 mAb. The positions of the beads are marked with asterisks. Insets are higher-magnified images of boxed areas. DIC, differential interference contrast. Scale bars: 10 µm. (C) Co-immunoprecipitation of endogenous PDGF receptor with nectin-3. The cell lysates of NIH3T3 cells treated with a chemical cross-linker DTSSP were immunoprecipitated with the anti-p85 pAb or normal rabbit IgG as a control, and were subjected to western blotting with the anti-nectin-3 pAb and the anti-PDGF receptor pAb. (D) Co-immunoprecipitation of PDGF receptor with nectin-3. Lysates of HEK293 cells transiently expressing PDGF receptor with or without FLAG-nectin molecules were immunoprecipitated with the anti-FLAG mAb. The immunoprecipitates were subjected to western blotting with the anti-PDGF receptor pAb and the anti-FLAG mAb. (E) No co-immunoprecipitation of EGF receptor with nectin-3 occurred. Lysates of HEK293 cells transiently expressing FLAG-nectin-3 with or without EGF receptor were immunoprecipitated with the anti-FLAG mAb, followed by western blotting as described in D. (F) Co-immunoprecipitation of PDGF receptor with nectin-3 through their extracellular regions. The cell lysates of HEK293 cells transiently expressing FLAG–nectin-3CP or FLAG–nectin-3EC with or without PDGF receptor were immunoprecipitated with the anti-FLAG mAb, followed by western blotting, as described in D. (G) Co-immunoprecipitation of PDGF receptor with nectin-3 independent of the phosphorylation of PDGF receptor. HEK293 cells transiently expressing PDGF receptor with or without FLAG–nectin-3 were incubated in the presence or absence of 50 µM AG1296, a PDGF receptor inhibitor, for 1 hour. Cell lysates were immunoprecipitated with the anti-FLAG mAb, followed by western blotting as described in D. The results shown in this figure are representative of three independent experiments.