Involvement of C-Jun N-terminal kinase/mitogen-activated protein kinase (JNK MAPK) in HIV-1 Tat-mediated expression of IL-8. (a-f) SVG astrocytes were pretreated with JNK MAPK inhibitor (SP600125) for 1 hour prior to transfection. (a, b) The expression levels of IL-6 and IL-8 at mRNA level were determined at 6 hours post transfection by real time RT-PCR. The values represented are normalized to their mock- transfected controls. (c, d) IL-6 and IL-8 protein concentrations in the cell culture supernatants at 48 hours post transfection were determined by multiplex cytokine assay. (e) Astrocytes were either mock-transfected or transfected with HIV-1 Tat plasmid for duration of 6 hours and p-JNK and JNK were measured in whole cell extracts. (f) The levels of p-c-jun were measured in whole cell extracts 6 hours after the transfection. (g) Astrocytes were transfected with JNK1 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 the panels (e), (f) and (g). 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.