FIG. 7.

Both NF-κB and IRF-1 are recruited to the promoter regions of genes involved in antigen processing and transport. (A and B) Results from a representative ChIP assay (A) and collective data from four experiments (B) showing the kinetics of the in vivo recruitment of p65, IRF-1, and RNA polymerase II to the bidirectional TAP1/LMP2 promoter following CD40 stimulation. ChIP assays were performed as described in detail in Materials and Methods. One-tenth of the volume of the chromatin obtained was used for PCR as input, and the remaining volume was immunoprecipitated with anti-p65 (α-p65), anti-IRF-1, or anti-RNA polymerase II (α-Pol II) antibody or subjected to mock (i.e., without antibody) precipitation. Precipitated DNA encompassing the TAP1/LMP2 promoter was then assayed by PCR. The quantification of the results was performed by measuring the intensities of the bands using the Tinascan version 2 software (B), and the levels of recruitment were expressed as percentages of recruited protein relative to the input. Lane 1 in panel A is a DNA molecular size marker, with the bands indicated by the asterisks corresponding to 500 bp. −, absent; 15′, 15 min. (C) Kinetics of the in vivo recruitment of p65 and IRF-1 to the promoter regions of the TAP2, LMP10, and tapasin genes. The data shown are the means of results from two independent experiments.