Figure 2.

cAMP represses RRAS gene expression mainly via CREB3. A) Luciferase reporter assays were performed to determine RRAS promoter activity in HUVECs in which cAMP effectors were silenced by siRNA for 48 h. Results are shown as means ± sem of 3 independent experiments performed in triplicate. ****P < 0.0001 (2-way ANOVA). B) Protein levels of R-Ras, PKA catalytic-α, CREB1, and ATF2 were determined by Western blot analysis. Knockdown of CREB3 was assessed by qRT-PCR due to lack of availability of good antibody for this purpose. Effect of cAMP effector silencing on R-Ras protein level was quantified by ImageJ software (bottom graphs). Arrow in upper band indicates ATF2. Ns, not significant. *P < 0.05, ****P < 0.0001 (2-way ANOVA).