FIGURE 6.

siRNA-mediated knockdown of AMPK or Rac1 and ADP-modulated eNOS phosphorylation. A, BAEC were transfected with control or AMPK siRNA, and 48 h later, cells were treated for 5 min with ADP (50 μm) or vehicle control. Lysates were resolved by SDS-PAGE, and immunoblots were analyzed by probing with the indicated antibodies. Equal loading was determined by probing for total eNOS, GSK3β, ACC, and ERK1/2, and AMPK knockdown was confirmed by probing for total AMPK. This blot is representative of four individual experiments that yielded equivalent results. Quantitative analysis of pooled data comparing relative eNOS Ser¹¹⁷⁹, eNOS Ser⁶³⁵, eNOS Ser¹¹⁶, and ACC phosphorylation is shown in B–E, respectively. F shows a representative immunoblot of lysates from BAEC prepared from cells transfected with control or Rac1 siRNA prior to ADP treatment (50 μm, 5 min). Protein knockdown was determined using an antibody directed against Rac1. The graphs to the right were obtained by quantitative digital chemiluminescence analysis of pooled data and represent relative phosphorylation of eNOS Ser¹¹⁷⁹ (G), eNOS Ser⁶³⁵ (H), and ERK1/2 (I), normalized to total eNOS or ERK where appropriate. These data are the result of four independent experiments that yielded similar results. * indicates p < 0.05, ** denotes p < 0.01, and *** represents p < 0.001. p, phospho; NS, not significant.