Fig. 2

PI3K/Akt and ERK signal pathways were involved in Tat-induced P2Y4R expression and inflammatory response in mouse astrocytes. a Primary mouse astrocytes were incubated in a serum-free medium overnight followed by treating with soluble Tat protein for different time point. Representative levels of phosphorylation of Akt (p-Akt) and p-ERK1/2 were displayed by Western blotting assay. These data are from three independent experiments. b, c Primary mouse astrocytes were incubated in a serum-free medium overnight followed by pretreating with or without PI3K pathway inhibitor LY294002 (10 μM) and ERK1/2 pathway inhibitor PD98059 (50 μM) for 2 h, respectively, prior to soluble Tat protein stimulus for 6 h. Representative levels of P2Y4R protein were determined by Western blotting assay. These data are from three independent experiments. d, e Primary mouse astrocytes were incubated in a serum-free medium overnight followed by pretreating with or without 50 μM PD98059 for 2 h, prior to soluble Tat protein stimulus for 6 h. d Relative mRNA and e release levels of TNFα, IL-6, IP-10, and MCP-1were measured by qPCR assay and Cytometric Bead Array, respectively. The data are from three independent experiments. The relative expression levels of genes were normalized to the expression of mouse Actin. *P < 0.05, **P < 0.01, and ***P < 0.001. f, g Primary mouse astrocytes were incubated in a serum-free medium overnight followed by pretreating with or without 10 μM LY294002 for 2 h, prior to soluble Tat protein stimulus for 6 h. f Relative mRNA and g release levels of TNFα, IL-6, IP-10, and MCP-1were measured by qPCR assay and Cytometric Bead Array, respectively. These data are from three independent experiments. The relative expression levels of genes were normalized to the expression of mouse Actin. The data are represented as the mean ± S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001