Fig. 5. ATM is upstream of the aphidicolin-stimulated Akt/CREB/eNOS/NO signaling pathway. (A) BAECs were treated with 20 µM aphidicolin or vehicle (DMSO) for 24 h, and the level of p-ATM-Ser
1981 was assessed using western blot analysis as described in
Fig. 1. (B-C4) After 100 nM siRNA specific for the ATM gene or scrambled siRNA was transfected into BAECs, the cells were treated with 20 µM aphidicolin or vehicle (DMSO) for 24 h, and then levels of eNOS, p-ATM-Ser
1981, p-Akt-Ser
473, or p-CREB-Ser
133 were measured using western blot analysis as described in
Fig. 1. (A-C4) Reprobing and quantitation of eNOS, p-ATM-Ser
1981, p-Akt-Ser
473, and p-CREB-Ser
133 was done as described in
Fig. 1. (D) After BAECs were prepared as described in
Fig. 5B, level of 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 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.
