Fig. 1. Aphidicolin increases NO production in BAECs with concomitant increase in expression and dimerization of eNOS protein and BH4 levels. (A) BAECs were treated with 20 µM aphidicolin or vehicle (DMSO) for the indicated times (0, 8, 24, or 48 h) and the level of NO production was electrochemically measured using an NO sensor (ISO-NOP, WPI), as described in the MATERIALS AND METHODS. (B) BAECs were treated with various doses of aphidicolin (0, 10, 20, or 40 μM) for 24 h and the level of NO production was measured as described in
Fig. 1A. (C) After BAECs were treated with 20 µM aphidicolin for the indicated times (0, 8, 24, or 48 h), total protein was obtained and the level of eNOS protein expression was detected using western blot analysis. Nitrocellulose membranes were re-probed using an anti-tubulin antibody to assess equal sample loading. (D) BAECs were treated with various doses of aphidicolin (0, 10, 20, or 40 μM) for 24 h and the level of eNOS protein expression was detected using western blot analysis as described in
Fig. 1C. (E) BAECs were treated with 20 µM aphidicolin or vehicle (DMSO) for 24 h, the eNOS dimer and monomer were separated using LT-PAGE, and their ratio was measured using western blot analysis as described in the MATERIALS AND METHODS. (F) In a separate experiment, BAECs were treated as described in
Fig. 1E and cellular BH4 level was measured using a BH4 ELISA kit (MyBioSource) as described in the MATERIALS AND METHODS. (C, D) Using western blot data, densitometry was done to quantitate eNOS protein expression relative to tubulin expression or (E) level of eNOS dimer relative to monomer. 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 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, ***
p<0.001.
