The cells were cultured with transfection mixture for 5 h, selleck kinase inhibitor and were then cultured in DMEM containing 10% FBS, 0. 5 ug mL LPS with or without different concentrations of resveratrol for 16 h. Luciferase activity of pNF B Luc and pRL TK constructs was measured sequentially using the Dual Luciferase Reporter Assay System. Variation in transfec tion efficiency was normalized by dividing the promoter construct activity by the respective co transfected pRL TK luciferase activity. Promoter activity of the NF B was expressed in units relative to values measured in cells cultured with control medium. Nuclear extract preparation and electrophoretic mobility shift assay Inhibitors,Modulators,Libraries Nuclear extracts were prepared as previously described. Protein concentration was determined using a Bio Rad protein assay kit with bovine serum albumin standards.

Activation of AP 1 was assayed by EMSA using a LightShift Chemiluminescent EMSA kit according to the manufactures instruc tion. Briefly, 6 ug of nuclear extract proteins were pre incubated with binding buffer for 5 min and then Inhibitors,Modulators,Libraries incubated with double stranded Inhibitors,Modulators,Libraries biotin labeled oligonucleotide containing consensus AP 1 bind ing site for 15 min at room temperature. For competition experiments, unlabelled oligonucleotides were added to the nuclear extracts at a 200 fold molar excess before the addition of the biotin labeled probe. DNA protein complexes were analyzed by electrophoresis in 4% poly acrylamide gels. Complexes were transferred to a nylon membrane and crosslinked to the membrane using a hand held UV lamp equipped with 312 nm bulbs.

Migration of the biotinylated oligonucleotides and their complexes was detected by chemiluminescence followed by exposure of the membrane to X ray films. Statistical analysis Data are presented as mean SD. All experiments were performed at least three times. Data were analyzed Inhibitors,Modulators,Libraries by a 1 way or 2 way ANOVA with a post hoc Bonferroni test. Differences were considered significant at p 0. 05. Results LPS induces proinflammatory cytokine and iNOS expression in microglia and astrocytes in which different signaling molecules are involved We first examined the effect of LPS on proinflammatory cytokine and iNOS expression by murine primary microglia and astrocytes, and explored the signaling molecules involved. As shown in Fig. 1, 0. 5 ug mL LPS significantly increased TNF a, IL 1b, IL 6, MCP 1 and iNOS mRNA levels in both cell types.

Activation of macrophages and microglia by LPS is thought to occur by binding of LPS to Toll like receptor 4, leading to activation of intracellular kinases and transcription factors like NF B and AP 1. We used inhibitors for MAP kinases and transcrip tion factors to determine whether Inhibitors,Modulators,Libraries activation of ERK1 2, HTC p38, JNK, NF B, or AP 1 contribute to the induction of proinflammatory cytokines and iNOS expression by LPS in microglia and astrocytes.