Fig. 5

Association of RelB and Tat binding to the HIV-1 LTR. a Schematic illustration of the HIV-1 LTR and location of oligonucleotide primers. ChIP primers for HIV-1 LTR analysis were designed to span the enhancer region, including the duplicated κB enhancers (− 104 to − 81) and specificity protein 1 binding sites (SP1, − 79 to − 47). Primers for analysis of initiated HIV-1 transcripts were directed against TAR. b–d TZM-bl cells were transfected with pFlag-Tat (6 μg) or vector plasmids. The relative expression of Tat and RelB in the transfected cells was monitored by a western blot assay (b). Chromatin of the fixed TZM-bl cells (1 × 10⁷) was immunoprecipitated with the indicated antibodies. Samples were assessed for enrichment of HIV-1 LTR DNA by PCR (c). Relative levels of the LTR DNA that were co-precipitated with RNA Pol II, RelB, and p52 were quantified by real-time PCR performed in triplicate. The data are normalized to the input controls. Data are representative of three independent experiments (d). e–h Control and RELB^−/− TZM-bl cells were transfected with pFlag-Tat (6 μg) or vector plasmids (e). The co-precipitated LTR DNA was assessed by PCR (f). Tat (g) and RNA Pol II (h) associated HIV-1 LTR was quantified by real-time PCR