Figure 3.

Effect of aggretin on VSMC PDGFR-β and Src phosphorylation. (A) VSMCs (1 × 10⁶ cells) were cultured in the presence of aggretin (0.1 µmol·L⁻¹) for various times and the reactions were stopped with lysis buffer. Immunoprecipitates (IP) of PDGFR-β were formed as described in the methods. VSMC lysates were applied to SDS-PAGE. Tyrosine-phosphorylated proteins were detected with Western blot (WB) using anti-phosphotyrosine mAb (4G10) coupled with ECL, and then reprobed with PDGFR-β mAb. (B) VSMCs (1 × 10⁶ cells) were cultured in the presence of aggretin (0.1 µmol·L⁻¹) for different times, and the reactions were stopped with lysis buffer. Src activation was detected with WB using anti-phospho-Src pAb (Tyr 216) coupled with ECL, whereas α-tubulin was taken as an internal control. Summary data of PDGFR-β and Src phosphorylation were presented (on the right) as mean density, as determined by a densitometer. Densitometric band intensities were normalized to static controls, and fold increases were calculated. The data are representative of at least three experiments. Data are presented as mean ± SEM (n = 4). *P < 0.05 as compared with control. ECL, enhanced chemiluminescence; PDGFR-β, platelet-derived growth factor receptor-β; SDS, sodium dodecyl sulphate; VSMC, vascular smooth muscle cell.