Figure 3. CREB contributes to Ang II-mediated increases in AT₁R, NF-κB, active Elk-1, c-fos.

Knockdown of CREB by siRNA in CATH.a cells. (A) RT-PCR and Western blotting (lower panel) showing efficient suppression of CREB mRNA in CATH.a cells. (B) Inhibitory effect of CREB siRNA on Ang II-induced p65 NF-κB activation. siRNA transfected cells were stimulated with Ang II (100 nM) for 1 hour. p65 NF-κB levels were detected by western blotting. (C) Effect of CREB siRNA on p65 NF-κB nuclear translocation. Cells were transfected with CREB siRNA and subjected to Ang II activation over 1 hour period. Following nuclear/cytoplasmic fractionation, the lysates were immunoblotted for p-CREB and p65 NF-κB levels by western blotting in both nuclear and cytoplasmic fractions. (D) CATH.a cells were transfected with anti-CREB siRNA followed by Ang II stimulation for 8 hours. The cells were then probed with specific antibody against pElk-1 and (E) p-cFos. (F) Cells were either mock transfected with vehicle or anti-CREB siRNA and subsequently stimulated with Ang II (100 nM). Following the specified time course, the cells were probed for AT₁R message (lower panel) and protein levels. Values are expressed as mean ± SEM. *p<0.05 vs control, #p<0.05 vs Ang II, †p<0.05 vs corresponding untreated nuclear fraction. n = 3–4 per treatment.