Figure 6

AEA action on MAPK activation is mediated through MEK-1 in Tat C cells, but not in Tat B cells. MEK inhibition by U0126 increases MAPK phosphorylation in Tat C + AEA, but to a lesser extent in Tat B + AEA. In both Tat B and Tat C cells, AEA induces peak phosphorylation at 2 hours (a, b). On exposure to AEA, MKP inhibition by Ro-318220 increase MAPK phosphorylation significantly more in Tat B cells (c) than in Tat C cells (d). Representative blots shown. Normalized levels of phosphorylated ERK1/2, JNK, p38 are shown as mean ± S.E (n = 3). *p < 0.05, **p < 0.01,***p < 0.001 vs. Control. Cropped Western blot data show silencing with MEK-1 or MKP-1 siRNA block MEK-1 or MKP-1 expression (e). Full-length blots are presented in Supplementary Fig. S6. On MKP-1 siRNA treatment of Tat B + AEA cells, there is suppression in TGF-β and IL-10. TNF-α, IFN-γ, IL-6, IL-12p70 and CXCL-10 are however elevated. Tat B + AEA treated cells with MEK-1 knockdown showed comparable levels of cytokines relative to Tat B + AEA only (f). MEK-1 siRNA in Tat C + AEA cells significantly reduced the levels of pro- inflammatory cytokines compared to Tat C + AEA only cells. Tat C + AEA cells with MKP-1siRNA showed similar profile to Tat C + AEA only cells (f). Treatment by pharmacological blockers for MKP-1 and MEK-1 in Tat B + AEA or Tat C + AEA cells show results similar to siRNA knockdown (g). ELISA was used to measure cytokine protein levels. Results shown are the mean ± S.E (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Tat B + AEA.