Recruitment of IRF1 to IL-12p35 and IL-4 promoters and their mRNA expression after exogenous IFN-γ stimulation. (A) Ex vivo PBMCs from HIV-S (n = 9, ▵) and HIV-R (n = 9, □) individuals were stimulated with exogenous IFN-γ (10 ng/mL). At the indicated time points, chromatin was isolated and immunoprecipitated with antibodies specific for IRF1 chromatin-immunoprecipitated DNA products were analyzed for the presence of IL-12p35 and IL-4 promoter using quantitative PCR. Quantitative PCR signals were normalized to input DNA. There was no difference in the levels of IRF1 binding between the unstimulated samples, cultured in media alone for 0, 60, or 180 minutes. Unstimulated sample from time = 0 is used as reference for calculating relative fold increases. (B) RNA were also isolated from the PBMCs of HIV-S (n = 10, ▵) and HIV-R (n = 11, □) individuals, after IFN-γ stimulation. IL-12p35 and IL-4 mRNA levels were examined using quantitative RT-PCR. There was no significant difference in mRNA levels between the unstimulated samples (time = 0, 20, 60, or 180 minutes). Unstimulated sample from time = 0 is used as reference for calculating relative fold increases. The RNA transcripts were normalized to endogenous 18S rRNA.