Involvement of NF-κB in HIV-Tat mediated up-regulation of IL-6 and IL-8. (a) SVG astrocytes were either mock-transfected or transfected with HIV-1 Tat plasmid and translocation of p65 was measured at 3, 6, 9 and 12 hours. Open bars and closed bars represent cytoplasm and nuclear fractions, respectively. (b) Primary astrocytes were treated with 200 ng/ml Tat protein and p-IκBα protein levels were measured from 0 minutes to 60 minutes. The bar graph represents the mean values obtained from two independent donors. (c-g) Astrocytes were pretreated with 10 μM concentration of NF-κB inhibitor (BAY11-7082) 1 hour prior to the transfection. The expressions of IL-6 and IL-8 were determined at 6 hours and 48 hours post transfection for mRNA (c, d) and protein (e, f), respectively. The values represented are normalized their mock-transfected controls. (g) Astrocytes were either mock-transfected or transfected with HIV-1 Tat plasmid for a duration of 6 hours and translocation of p65 was measured. (h-k) Astrocytes were transfected with siRNA (scrambled or p65 or p50) for 48 hours, followed by transfection with HIV-1 Tat plasmid. The expression of IL-6 and IL-8 was determined at 6 hours and 48 hours post transfection for mRNA (h, i) and protein (j, k), respectively. Each experiment was done at least in triplicate and each bar represents the ± SE of three individual experiments. Statistical analyses was performed by one-way ANOVA and ** denotes P-value of ≤ 0.01.