Fig. 4. Akt mediates eNOS promoter transactivation by inducing p-CREB-Ser
133. (A-B2) BAECs were treated with 20 µM aphidicolin or vehicle (DMSO) for 24 h in the absence or presence of 10 µM H-89, a PKA inhibitor, and then, levels of eNOS protein and p-CREB-Ser
133 were assessed using western blot analysis as described in
Fig. 1. (C-D3) BAECs were treated with 20 µM aphidicolin or vehicle (DMSO) for 24 h in the absence or presence of 5 µM LY294002, a phosphoinositide 3-kinase inhibitor, and then, levels of eNOS protein, p-Akt-Ser
473, and p-CREB-Ser
133 were detected using western blot analysis as described in
Fig. 1. (A-D3) Reprobing and quantitation of eNOS, p-Akt-Ser
473, or p-CREB-Ser
133 were done as described in
Fig. 1. (E) After BAECs were treated with 20 µM aphidicolin or vehicle (DMSO) for 24 h in the absence or presence of 5 µM LY294002, NO release was measured as described in
Fig. 1. (F) In a separate experiment, after cDNA encoding dn-Akt or an empty vector was transfected into BAECs, the cells were treated with 20 µM aphidicolin or vehicle (DMSO) for 24 h and then, NO production was measured as described in
Fig. 1. All experiments were performed at least four times independently and blots shown are representative of at least four experiments (n=4). Bar graphs depict mean fold alterations above/below the controls (± SD). Statistical significance was evaluated using ANOVA. All differences were considered to be statistically significant at a
p value of <0.05.
