FIG 3.

R6 competes with IRF3 for binding to CBP. (A) Telomerized RFs were transfected with pcDNA3.1-R6HA (5, 10, or 20 μg DNA) or empty pcDNA3.1 for 40 h and subsequently transfected with poly(I · C) for 6 h. Nuclear lysates were immunoprecipitated with anti-CBP antibody and then subjected to SDS-PAGE and probed with anti-HA antibody or anti-pIRF3 antibody. The nuclear lysates were probed for HA expression, with PARP as a loading control and a control for purity of nuclear fractionation. (B) EMSA was performed on whole-cell extracts (20 μg) derived from telomerized RFs transfected with pcDNA3.1-R6HA or empty pcDNA3.1. The biotin-labeled probe corresponds to the PRDI-PRDIII motif (5′-GAAAACTGAAAGGAGAACTGAAAGTG-3′) of the IFN-β promoter. Anti-CBP antibody and anti-IRF3 antibody were added as indicated to demonstrate the presence of CBP and IRF3 in the DNA-protein complexes. For oligonucleotide competition, a 500-fold molar excess of unlabeled PRDI-PRDIII probe was added as indicated. (C) Telomerized RFs were pretreated with MG132 or left untreated. The cells were then transfected with pcDNA3.1-R6HA or empty pcDNA3.1 for 40 h and then transfected with poly(I · C) for 6 h. Whole-cell lysates were immunoprecipitated with anti-IRF3 or anti-TBK and then subjected to SDS-PAGE and probed with anti-IRF3 or anti-TBK to gauge total levels of IRF3 and TBK within the cells. The nuclear lysates were subjected to SDS-PAGE and probed with anti-pIRF3 antibody or PARP, which served as a loading control and a control for purity of nuclear fractionation.