Effect of anti–integrin α₂mAb and VEGF mAb on aggretin-induced Akt and ERK phosphorylation, and VEGF expression by aggretin. HUVECs (1 × 10⁶ cells) were pretreated with anti–α₂ integrin mAb, A2-IIE10 (50 μg/mL; A), or VEGF mAb (1 μg/mL; B) for 30 minutes, then cultured in the presence of 0.1 μM aggretin for various times, and then lysed with lysis buffer. Akt and ERK activation was detected with Western blotting using antiphospho-Akt or antiphospho-ERK mAb coupled with ECL, whereas α-tubulin was the internal control. The pattern shown is a representative one of at least 3 similar results. (C) HUVECs (1 × 10⁶ cells) were pretreated with PBS or anti–integrin α₂ mAb, A2-IIE10 (50 μg/mL), for 30 minutes, and then cultured in 0.1 μM aggretin for 6 hours or 24 hours. Cells were lysed with lysis buffer and detected with Western blotting using anti-VEGF mAb coupled with ECL, whereas α-tubulin was the internal control. Quantitative analyses of VEGF production were presented as mean density as determined by densitometer. Densitometric band intensities were normalized to static controls, and fold increases were calculated. Data are presented as mean ± SEM (n = 3). *P < .05; **P < .01 as compared with that of control. The pattern shown is a representative one of at least 3 similar results. (D) HUVECs (1 × 10⁶ cells) were cultured in presence or absence (CTL) of 0.1 μM aggretin for 3, 6, or 24 hours. VEGF levels were determined by ELISA, as described in “Materials and methods.” Data are means ± SEM of 3 experiments.