FIGURE 2.

CaMKKβ inhibition or down-regulation impairs VEGF-induced ACC phosphorylation but not eNOS phosphorylation. HUVEC were either preincubated for 30 min with STO-609, a CaMKK inhibitor, at the indicated concentrations (A, C, and E) or pretreated with CaMKKβ-specific or control-siRNA (1 μg/30-mm-diameter dish, 72 h) (B, D, and F) and subsequently stimulated with 50 ng/ml VEGF for 5 min (A, B, E, and F) or 20 min (C and D). A, B, E, and F, cells were lysed and subjected to immunoblotting using antibodies against phosphorylated eNOS (serine 1177) or total eNOS and against phosphorylated ACC or total ACC. Typical experiments and the densitometric analysis of three experiments for each staining are shown (mean ± S.E.). +, p < 0.05 versus unstimulated controls; *, p < 0.05 versus non-inhibitor-treated VEGF-stimulated cells. C and D, cells were denaturated with ethanol and processed for cGMP formation. cGMP was determined in ethanolic extracts by means of radioimmunoassay and calculated as pmol/mg cell protein, which was assayed in parallel samples (mean ± S.E., n = 4). +, p < 0.05 versus unstimulated controls.