FIGURE 8.
CaMKIIδ inhibits CREB binding to Sik1 and Rgs2 promoters. A, ChIP were performed using anti-CREB antibody on unstimulated VSM cells. CREB-binding elements in Sik1 and Rgs2 promoters were amplified by quantitative PCR and products resolved on an agarose gel as follows: from sheared chromatin prior to IP (input), chromatin immunoprecipitations (IPs) (IP CREB), and IP (IgG) control. B–D, effect of CaMKII mutant expression on thrombin-stimulated CREB binding to SIK1 (C) and RGS2 (D) promoters. VSM cells were transduced with adenovirus encoding dominant-negative (DN)-CaMKII (100 multiplicity of infection), constitutively active CA-CaMKII (50 multiplicity of infection), or adenovirus control (Ad-virus). Thrombin (50 nm) was added 10 min prior to fixation and processing for ChIP. DN-CaMKII and CA-CaMKII constructs were detected by immunoblotting with anti-HA antibodies (immunoblot (IB): HA) in supernatants following ChIP (B). Ct values were normalized to thrombin stimulation alone. Values shown are mean ± S.E., n = 3, and analyzed by one-way ANOVA. *, p < 0.05; **, p < 0.01, by Dunnett's post hoc test.