FIGURE 5.

18% CS induced PECAM1 tyrosine phosphorylation and internalization. HPAEC monolayers were stimulated with 18% CS for indicated periods of time or left under static conditions. A, 18% CS-induced PECAM1 tyrosine phosphorylation was analyzed by immunoprecipitation (IP) assays with PECAM1 antibody followed by probing for phosphotyrosine. The results are representative of three independent experiments. B, PECAM1 distribution in control and stretched EC was examined by immunofluorescence staining. The bar graph represents quantitative analysis of PECAM1 peripheral accumulation. The data are expressed as means ± S.D. of three independent experiments; *, p < 0.05 versus ns-RNA. C, cell surface proteins were labeled with Sulfo-NHS-SS-Biotin. Biotinylated proteins were collected using streptavidin-agarose, and PECAM1 content was evaluated by Western blot analysis with corresponding antibodies. D, PECAM1 immunoprecipitation under nondenaturing conditions was performed from control EC or cells subjected to 18% CS for 15 min. The presence of VEGFR2 and VE-cadherin in immune complexes was examined by Western blot. E, PECAM1 and VE-cadherin co-localization in lung EC was examined by double immunofluorescence staining for PECAM1 (red) and VE-cadherin (green). Insets, higher magnification images of peripheral cell areas marked by quadrangles.