Figure 5. Tat activates the p65-dependent expression of NF-κB-responsive genes by occupying the NF-κB enhancers and promoting the p65 recruitment with IκB-α displacement. HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, p3xFLAG-Tat C(22,25,27), p3xFLAG-Tat R(49–57) or empty vector (10 µg); for p65 RNA interference, cells (5 × 106) were transfected with p3xFLAG-Tat (10 µg) and siRNA p65 or siRNA control (200 pmol). Twenty-four-hour post-transfection, total RNA was extracted and analysed by real-time PCR to evaluate the expression of MIP-1α (A), CSF3 (B), LTA (C), NFKBIA (D), TLR2 (E), GAPDH (F), ACTB (G) genes. (H) HeLa cells (5 × 106) were transfected with p3xFLAG-Tat, or empty vector (10 µg) and 48 h later ChIP was performed with anti-p65 antibody. Real-time PCR was performed with primers specific for the indicated promoters. (I) HeLa cells (5 × 106) were transfected with empty vector, or p3xFLAG-Tat (10 µg) in presence or absence of siRNA p65, or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-FLAG antibody. Real-time PCR was performed with primers specific for the indicated promoters. (J) HeLa cells (5 × 106) were transfected with empty vector, p3xFLAG-Tat (10 µg), or empty vector plus siRNA IκB-α or siRNA control (200 pmol); 48 h later, ChIP was performed with anti-IκB-α antibody. Real-time PCR was performed with primers specific for the indicated promoters. Values (mean ± SE, n = 3) are shown. The asterisks indicate statistically significant differences compared to the control (empty vector) according to the Student's t-test (P < 0.05).