Figure 5. CCTη regulates p65 transcriptional activity by modulating CBP-dependent p65 acetylation of K122 and K123.

A: HEK293 cells were transfected with CBP, pCDNA3 (control) or myc-p65 and scrambled (sc) or CCTη shRNA, incubated for 3 days and p65 was immunoprecipitated using c-myc-specific agarose beads. Acetylated p65 (Ac-p65) and total p65 were detected using antibodies to acetylated lysine or p65, respectively. B: HEK293 cells were transiently transfected with scrambled (sc) or CCTη shRNA, incubated for three days and endogenous acetylated CBP (Ac-CBP) and total CBP were detected using antibodies to acetylated lysine or CBP after immunoprecipitation, respectively. C: HEK293 cells were transfected or not with scrambled (sc) or CCTη siRNA, stimulated with TNF (10ng/ml) for indicated times and nuclear extracts were prepared. CCTη was immunoprecipitated using TCP-1η antibody. Unrelated EP-1 antibody was used as a goat IgG control (*). D: Rela ^−/− MEF reconstituted with either WT p65, K310R, K221R or K122/123R mutant were exposed to scrambled (sc) or CCTη siRNA for three days and stimulated with TNF (10 ng/ml) for indicated times. Cxcl10 mRNA levels were analyzed by qPCR as described under “Materials and Methods”. Data represent mean relative mRNA levels ± SEM (n≥3). CCTη and p65 were detected in cell extracts by western blotting (representative of three independent experiment).