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Figure 3 | Journal of Neuroinflammation

Figure 3

From: 5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability

Figure 3

ETYA acts in a PPAR-α-independent manner to suppress CCL2/MCP-1 expression through induction of MKP-1 expression and phosphatase activity. (A and B) ETYA- or WY-14643-treated astrocytes were stimulated with IFN-γ for 2 h. MKP-1 protein levels and JNK phosphorylation were analyzed by Western blotting (A), and MKP-1 and CCL2/MCP-1 transcript levels were determined by qRT-PCR (B). (C and D) Cell lysates immunoprecipitated with an anti-MKP-1 antibody were incubated with p-NPP for 4 h and then analyzed spectrophotometrically at 405 nm (C), or incubated with lysates from IFN-γ-activated astrocytes followed by Western blot analysis of eluates (D). (E-I) Primary astrocytes were transfected with an MKP-1-specific (E and F) or PPAR-α-specific siRNA duplex (G-I) or a nonsilencing control siRNA. Forty-eight h after transfection, cells were stimulated with IFN-γ for 2 h or 12 h in the absence or presence of ETYA. MKP-1 protein levels and phospho-JNK, and transcript levels of MKP-1 and CCL2/MCP-1 were determined by Western blot analysis (E and G) and qRT-PCR (F and H). MCP-1 protein secretion was analyzed by ELISA (I). Efficiency of siRNA-mediated PPAR-α silencing was demonstrated by monitoring expression of acetyl CoA synthase (ACS), a PPAR-α-dependent gene (data not shown). Values are means ± SDs. of three independent experiments. *p < 0.05, ** p < 0.01, NS; non significant.

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