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. 2005 Dec 1;25(1):139–149. doi: 10.1038/sj.emboj.7600900

Figure 5.

Figure 5

NF-κB1 p50 inhibits basal HIV expression in latently infected T cells by excluding RNA Pol II. (A) Stable shRNA knockdown of NF-κB1 p50 in latently infected T cells. Parental J-Lat 6.3 cells or stably transfected clones expressing an shRNA vector directed against a scrambled sequence or NF-κB1 p50 were selected and lysates were analyzed for p50 or control β-tubulin expression. (B) Fixed chromatin extracts from J-Lat 6.3 cells stably transfected with scrambled control shRNA or anti-NF-κB1 p50 shRNA treated with 20 ng/ml TNF-α for 30 min or left untreated were immunoprecipitated with the indicated antibodies. Samples were analyzed for enrichment of HIV LTR DNA by UV visualization of PCR products on an ethidium bromide-stained agarose gel. Specific enrichment was quantitated by real-time PCR; mean of three measurements is indicated beneath each band image. (C) Cells stably transfected with control- or NF-κB1 p50-shRNA were treated with 100 nM TSA for 2 h or left untreated. Total RNA was extracted, and initiated or elongated HIV RNA transcripts were quantitated by real-time RT–PCR. Bars represent the mean of triplicate samples; error bars represent standard deviation. Data are representative of three independent experiments.