Fig. 2.

Effect of inhibitors of PLC and CaMKK on VEGF-stimulated AMPK activity. HAECs were incubated in the presence or absence of 10 ng/ml VEGF for 5 min after preincubation with 100 nM wortmannin, 1 μM PP1, 10 μM U73122 or 25 μM STO-609 for 45 min and lysates prepared. (A) Total AMPK was immunoprecipitated from HAEC lysates and assayed for AMPK activity. The results are expressed as the means ± SD % basal AMPK activity for four independent experiments. *p < 0.01 relative to value in absence of inhibitor. (B) AMPK immunoprecipitates were resolved by SDS–PAGE, transferred to nitrocellulose and probed with the antibodies indicated. Representative immunoblots are shown, repeated with similar results on four different samples of lysates. (C) HAEC lysates were resolved by SDS–PAGE, transferred to nitrocellulose and probed with anti-CaMKKα antibodies. CaMKKβ was immunoprecipitated from HAEC lysate, subjected to Western blotting and probed with anti-CaMKKβ antibodies. A lysate prepared from whole rat brain was used as a positive control. Representative immunoblots are shown.