Figure 3

Resistin increased neuroinflammation through its binding to TLR4. (Panel A) Immunoprecipitation/Immunoblot (IP/IB) analysis of the direct association of resistin with TLR4 in protein extracts from SH-SY5Y cells treated with resistin (100 ng/ml) for 16 h in the presence or absence of the cross-linker agent BS3, the shown blots are from the same membrane but blotted with different antibodies following immunoprecipitation with anti-TLR4 antibody. (Panel B,C) SH-SY5Y cells were treated with or without resistin and then Akt and p38 MAP kinase phosphorylation measured by Western blots using adequate antibodies and the presented blots was from the same membrane but blotted successively with different antibodies, the band densities were quantified and expressed as phosphorylated/total proteins. Data were presented as means ± SEM (n = 3), * and *** denoted significant differences vs control at p < 0.05 and p < 0.0005, respectively. (Panel D) human SH-SY5Y neuroblastoma cells stably transfected with NF-κB luciferase reporter gene were treated resistin and relative luciferase activity determined. (Panel F) Human SH-SY5Y neuroblastoma cells were treated with resistin in the presence or absence of Akt, Erk, JNK or p38 MAP kinase inhibitors, and then IL6 and TNFα expression was determined using specific primers for these two genes, and normalized using GAPDH. Data were presented as means ± SEM (n = 3). Columns marked by different letter differ (with or without asterix concerns TNFα or IL6, respectively) significantly (p < 0.05). For other panels, data were presented as means ± SEM (n = 3), * and *** denoted significant differences at p < 0.05 and p < 0.005, respectively. (Panel E) Human SH-SY5Y neuroblastoma cells were treated with resistin or placebo and then IL6 and TNFα expression was determined using adequate primers, and normalized using GAPDH. Data were presented as means ± SEM (n = 3), * and *** denoted significant differences at p < 0.05 and p < 0.001, respectively. (Panel F).