Effect of aggretin on PI3K p85α. (A) HUVECs (1 × 106 cells) were cultured in the presence or absence of aggretin (0.1 μM) for various times and then lysed with lysis buffer. After immunoprecipitation (IP) of PI3K p85α, HUVEC lysates were applied to SDS-PAGE (8%). Tyrosine-phosphorylated proteins were detected with Western blotting (WB) using antiphosphotyrosine mAb (4G10) coupled with ECL, and then reprobed with polyclonal antibody (pAb) against PI3K p85α. (B) HUVECs (1 × 106 cells) were cultured in the presence of various concentrations of aggretin for 30 minutes or 24 hours, and then lysed with lysis buffer. Immunoprecipitation and Western blotting were used to measure the tyrosine phosphorylation induced by aggretin, and then reprobing was performed with pAb against PI3K p85α as an internal control. The pattern shown is a representative one of at least 3 similar results.
