Figure 5
PECAM-1 interacted with Pyk2 physically in vivo. A and B Binding of endogenous PECAM with endogenous Pyk2 in H460 (A) and A549 (B) cells. Confluent H460 and A549 cells from regular cell culture dishes were plated onto 100-mm polyHEMA-coated dishes for 3 or 15 h. Cells were harvested and lysed. Equal amounts of whole-cell extracts were immunoprecipitated with anti-PECAM-1 antibody, anti-Pyk2 antibody, or anti-Flag antibody followed by immunoblotting with anti-Pyk2 antibody or anti-PECAM-1 antibody. C, In vivo interaction of Pyk2 and PECAM-1 in 293T cells. 293T cells (1 × 106/100-mm dish) were co-transfected with pCDNA3.1 empty vector, PECAM FL construct, PECAM Mu (Δ11-16) construct, PYK2 construct, or a combination as indicated. After treatment for 48 h, equal amounts of whole-cell extracts were immunoprecipitated with anti-PECAM antibody and immunoblotted for Pyk2 and PECAM (upper) or immunoprecipitated with anti-Pyk2 antibody and immunoblotted for PECAM and Pyk2 (middle). Western blot analysis of equal amounts of cell extracts was also performed in parallel (lower). FL: Full-length; Mu: Mutant (Δ11-16).