Involvement of p38 mitogen activated-protein kinase (MAPK) in the induction of IL-6 and IL-8 by HIV-1 Tat. (a-f) SVG astrocytes were transfected with siRNA (scrambled or p38α or p38β or p38γ or p38δ) for 48 hours. Then they are mock-transfected or transfected with plasmid encoding HIV-1 Tat. (a, b) The expression levels of IL-6 and IL-8 at mRNA were determined by real time RT-PCR at 6 hours post transfection. The values represented are normalized to their mock-transfected controls. (c, d) The protein concentrations of IL-6 and IL-8 were determined in the cell culture supernatants at 48 hours post transfection by multiplex cytokine assay. (e) Astrocytes were transfected with p38β siRNA for 48 hours prior to transfection with HIV-1 Tat plasmid. The translocation of p65 is measured 6 hours after transfection. Open bars and closed bars represent cytoplasm and nuclear fractions, respectively. (f) Astrocytes were transfected with p38δ siRNA for 48 hours, followed by transfection with HIV-1 Tat plasmid. The levels of p-c-jun were measured after 6 hours of transfection. A representative Western blot is shown in panels (e) and (f). 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.