Figure 3.

Impaired transcriptional activity of RRPA mutant protein and the underlying mechanism. (A) Transactivation by RRPA p65 detected by reporter gene assay in MEFs. MEFs were transfected with pBIIx-luc and control construct Renilla for 24 h; stimulated with TNFα, LPS, or IL-1 (10 ng/mL) for 6 h; and analyzed. (B) The ability of p65 to interact with CBP was determined by immunoprecipitation. Immunoblotting for IκBα and nuclear p65 was performed to confirm the stimulation by LPS. (C) The ability of p65 to interact with HDAC3 was detected by immunoprecipitation. MEFs were stimulated with TNFα. (D) ChIP assay was performed with untreated or TNFα-treated (2 h) MEFs using the indicated antibodies. Precipitated IL-6 κB site DNA and IκBα κB site DNA were assayed by semiquantitative PCR. (E) Differential expression of NF-κB-regulated genes in MEFs. Total RNA isolated from unstimulated MEFs or MEFs stimulated with TNFα for 1, 3, or 5 h was quantified by real-time PCR and normalized to the level of GAPDH.