FIGURE 5.

Effects of siRNA-mediated knockdown of PKA and CaMKKβ on ADP signaling in endothelial cells. A, 48 h after BAEC were transfected with control or PKA siRNA, cells were treated with 50 μm ADP for 5 min. Cell lysates were resolved by SDS-PAGE, and immunoblots were probed using the indicated antibodies. Equal loading was confirmed by probing for total eNOS, AMPK, GSK3β, and ERK1/2. siRNA-mediated knockdown was determined by probing with antibodies for total PKA. Shown is an immunoblot representative of three individual experiments that yielded equivalent results. B and C show quantitative analysis of pooled data for relative phosphorylation of eNOS Ser¹¹⁷⁹ and eNOS Ser⁶³⁵, respectively. D, lysates were prepared from cells transfected with control or CaMKKβ siRNA and then treated for 5 min with ADP (50 μm). A representative immunoblot is shown here with knockdown confirmed by probing for total CaMKKβ. Quantitation of relative phosphorylation of eNOS Ser¹¹⁷⁹ (E), eNOS Ser⁶³⁵ (F), eNOS Ser¹¹⁶ (G), AMPK (H), and ACC (I) was assessed using digital chemiluminescence analysis of pooled data. Each bar in the graph represents the mean value ± S.E. of four independent experiments that yielded similar results. * indicates p < 0.05, ** indicates p < 0.01, and *** denotes p < 0.001. p, phospho; NS, not significant.