Fig. 1

Reduced phosphorylation of TBK1, IRF3, and c-Jun in bid-deficient glia and macrophages compared with wt. a, b wt and bid-deficient microglia were stimulated with LPS (100 ng/ml) for 1 h and lysed in RIPA buffer, and phosphorylated TBK1 levels were determined by Western blot (n = 6–7, 6–7 cultures from 6 separate platings, p = 0.0068, Kruskall-Wallis, Dunn’s non-parametric multiple comparison post hoc test). c, d wt and bid-deficient microglia were stimulated with LPS (100 ng/ml) for 5, 15, and 30 min or 1 h and lysed in RIPA buffer, and phosphorylated-IRF3 levels were determined by Western blot (n = 3, 3 cultures from 3 separate platings, p = 0.041, paired two-tailed t test, wt Veh vs. LPS 1 h). e, f wt and bid^−/− macrophages were stimulated with PolyI:C (100 ng/ml) for 30 min or 1 h and lysed in RIPA buffer, and phosphorylated-TBK1 levels were determined by Western blot (n = 4–6, 4–6 cultures from 5 separate platings, p = 0.0006 one-way ANOVA, Tukey’s multiple comparison post hoc test). g, h wt macrophages were transfected with an siRNA targeting Bid. Forty-eight hours post transfection, when Bid is optimally silenced, the cells were stimulated with LPS (100 ng/ml) for 1 h and lysed in RIPA buffer, and phosphorylated-TBK1 levels were determined by Western blot (n = 3, 3 cultures from 2 separate platings, p = 0.0588, paired two-tailed t test siControl LPS 1 h vs. siBid LPS 1 h). i, j wt and bid-deficient microglia were stimulated with LPS (100 ng/ml) for 5, 15, and 30 min or 1 h and lysed in RIPA buffer, and phosphorylated c-Jun levels were determined by Western blot (n = 5–7, 5–7 cultures from 6 separate platings, p < 0.0001, one-way ANOVA, Tukey’s multiple comparison post hoc test). k, l wt and bid-deficient macrophages were stimulated with LPS (100 ng/ml) for 30 min or 1 h and lysed in RIPA buffer, and phosphorylated c-Jun levels were determined by Western blot (n = 5–7, 5–7 cultures from 5 separate platings, p = 0.0017 one-way ANOVA, Tukey’s multiple comparison post hoc test). All samples were lysed in RIPA buffer containing protease and phosphatase inhibitors. The protein levels were quantified using optical densities. The data are represented as mean ± SD