Figure 6. Involvement of p38 MAPK pathway in Nef-mediated IL-6 and IL-8 increase.

SVGA astrocytes were pretreated with 10 μM of specific p38α and p38β inhibitor SB203580 (A–D) or transfected with siRNA against p38 isoforms (E–H) prior to transfection with nef-encoding plasmid. The expression levels of IL-6 and IL-8 mRNA and protein were determined at 6 h and 48 h post-transfection by real-time RT-PCR (A, C, E and F) and multiplex cytokine assay (B, D, G and H), respectively. In separate experiments, 7 × 10⁵ SVGA astrocytes were seeded in 6-well plates and pre-treated with SB203580 (10 μM) or transfected with siRNA against p38β prior to transfection with a plasmid encoding HIV-1 Nef (I–J). The cells were harvested at 3 h for isolation of cytoplasmic and nuclear proteins followed by western blotting for determination of p65 expression levels in the cytosol and nucleus. The figure shows one set of data that is representative of 3 independent experiments. The percentage expression of p65 was calculated relative to the nef-transfected wells at 3 h which was considered 100%. GAPDH was used as a loading control for cytoplasmic extracts and Lamin B was used as a loading control for nuclear extracts. The values represented for mRNA are relative to the mock-transfected controls. Each of the bars represents mean ± SE of three (A–D and I–J) or five (E–H) independent experiments in triplicates. Statistical analysis was performed using one-way ANOVA with an LSD post-hoc test t in which * represents p-value ≤ 0.05 and ** represents p-value ≤ 0.01.