Fig. 3. CREB mediates aphidicolin-stimulated eNOS expression. (A) After the A-CREB construct, a dominant-negative (dn) inhibitor of CREB, or empty vector was transfected into BAECs, cells were incubated for 24 h in the absence or presence of 20 µM aphidicolin. Levels of eNOS and CREB protein expression were measured using western blot analysis as described in
Fig. 1. (B) In a separate experiment, after the A-CREB construct or empty vector was transfected into BAECs, the eNOS promoter pGL2-eNOS (−1600) fused into a luciferase reporter plasmid was transfected into the cells. The cells were treated with 20 µM aphidicolin or 20 µM forskolin for 24 h, and an eNOS promoter assay was performed as described in
Fig. 2. (C) BAECs were treated with various doses of aphidicolin (0, 10, 20, or 40 μM) for 24 h and the level of p-CREB-Ser
133 expression was detected using western blot analysis as described in
Fig. 1. (A, C) Reprobing and quantitation of eNOS or p-CREB-Ser
133 relative to tubulin or CREB expression, respectively, were conducted 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 either Student’s
t test or ANOVA. All differences were considered to be statistically significant at a
p value of <0.05. **
p<0.01.
