Figure 4.

SHP-2 interacts with ICAM-1 and the VE-cadherin–β-catenin complex. A) EA.hy926 cells that were transfected with mouse ICAM-1 cDNA were crosslinked and lysed. Immunoprecipitation (IP) of proteins from cell lysates with Abs to SHP-2 was performed, followed by Western blot analysis with anti–SHP-2, anti–ICAM-1, anti–β-catenin, and anti–VE-cadherin Abs. Total protein levels of ICAM-1, SHP-2, β-catenin, and VE-cadherin were also determined by Western blot. B) EA.hy926 cell were treated as in panel A, followed by IP with Ab to VE-cadherin and Western blot analysis with anti–ICAM-1 and anti–VE-cadherin Abs. Total protein levels of ICAM-1 and VE-cadherin were determined by Western blot. C) EA.hy926 cell were cotransfected with mICAM-1 cDNA and SHP-2 siRNA or control (Ctrl) siRNA, and freshly isolated and activated neutrophils were added to EA.hy926 cell for 2 h, then cells were washed twice and lysed. Cell lysates were immunoprecipitated with Ab to VE-cadherin and Western blot analysis with anti–ICAM-1 and anti–VE-cadherin Abs. Then, ICAM-1–VE-cadherin expression was quantitated. Total protein levels of ICAM-1 and VE-cadherin were also determined by Western blot. In panels A–C, 3 independent experiments were performed. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Data are presented as means ± sem, and data analysis was performed by Bonferroni post-tests after 2-way ANOVA in panel C. ***P < 0.001.