Effects of sCD40L on eNOS mRNA levels in HCAECs. (A) Concentration-dependent study. HCAECs were treated with different concentrations of sCD40L for 24 hours. The eNOS levels were determined by real-time PCR. Heat-inactivated (HI) sCD40L was included as a negative control. (B) Time course study. HCAECs were treated with sCD40L (5 μg/mL) for different times. The eNOS levels were determined by real-time PCR. (C) Effect of anti-CD40L antibody and (D) effect of anti-CD40 antibody. HCAECs were pretreated with different concentrations of anti-CD40L antibody or anti-CD40 antibody for 30 minutes and followed with sCD40L treatment for 24 hours. The eNOS mRNA levels were determined by real-time PCR. Isotype IgG was used for a negative control. (E) eNOS mRNA stability. HCAECs were treated with actinomycin D (2.5 μg/mL) in the presence or absence of sCD40L (5 μg/mL) for different time points, and eNOS mRNA levels were determined by real-time PCR. (F) eNOS mRNA 3′UTR-binding molecules. Biotin-labeled human eNOS mRNA 3′UTR probe was incubated with cytoplasmic extracts of HCAECs treated with or without sCD40L for 24 hours. The binding reaction was electrophoresed on a native polyacrylamide gel. For the competition experiment, excess unlabeled RNA probe was preincubated with cytoplasmic protein prior to the addition of biotin-labeled RNA probe. *P < .05 and **P < .01, compared with the control. #P < .05 and ##P < .01, compared with sCD40L treatment. n = 3. Data are means and SE of multiple experiments (n).