Figure 3

JAK2 is phosphorylated and associates with TNFR1 in response to TNFα treatment. TNFα receptor (TNFR1) was immunoprecipitated from cell lysates of glia that had been treated with TNFα (5 ng/ml) for 0, 5 or 10 minutes, and western-blotted using a phospho-JAK2 specific antibody. Incubation of glial cells with TNFα stimulated a rapid association between phosphorylated JAK2 and TNFR1 (A). TNFα treatment of glial cells also induced phosphorylation of STAT1 at 10 minutes (B). The TNFα-stimulated phosphorylation of JAK2 and STAT1 was attenuated when cells were co-incubated with SAR317461 (C). Representative immunoblots of three separate experiments are shown. Incubation of glial cells with TNFα (5 ng/ml) stimulated release of IL-6 at 6 h (D, ^*** P < 0.001; ANOVA; n = 3), an effect that was inhibited when cells were co-treated with TNFα and TG101209 (D, ⁺⁺⁺ P < 0.001; ANOVA; n = 3). TNFα had no adverse effect on cell viability after 24 h of treatment (E, *P < 0.05; Student’s t-test for independent means; n = 3).