Fig. 1

TLR5 is constitutively expressed in microglia, and its activation triggers PDK1 and Akt phosphorylation in a PI3K-dependent manner. a Relative TLR5 expression levels were assessed in primary neonatal microglia, primary adult microglia, primary astrocytes, and primary cortical neurons isolated from C57BL/6 mice, as well as Oli-neu cells by quantitative RT-PCR (fold-change compared to neurons). TATA sequence binding protein (TBP) was used as housekeeping control (n = 3). Results are represented as mean ± SD. Data were analyzed by one-way ANOVA followed by Newman-Keuls test. *P < 0.05; **P < 0.01. b Cultured neonatal microglia, astrocytes, and neurons from C57BL/6 (WT) mice, as well as microglia from Tlr5^−/− mice were stained with antibody directed against TLR5 and co-stained with Iba1, S100β, or NeuN antibody serving as microglial, astrocyte, and neuronal marker, respectively. Scale bar, 10 μm. c Western blot analysis of microglial lysates using antibodies against p-Akt and total Akt after incubation of microglia with 100 ng/ml flagellin (FLA) for 0, 5, 15, 30, or 60 min (n = 3). Representative blots are shown in the upper panel. The graph depicts the average intensity ratio of the bands compared to control (lower panel). Data are expressed as mean ± SEM and were analyzed by one-way ANOVA followed by Dunnett’s post hoc test. **P < 0.01 vs. control. d Western blot analysis of FLA-mediated Akt phosphorylation after treatment of microglia with LY294002 (25 and 50 μM), Wortmannin (0.1 and 1 μM), and anti-mTLR5-IgG (1 μg/ml) (upper panel, n = 3). The graph shows the average intensity ratio of the bands compared to control (lower panel). e Western blot analysis of microglial lysates with antibodies against p-PDK1 and β-actin after incubation of microglia with 100 ng/ml FLA, FLA plus LY294002 (25 and 50 μM), FLA plus Wortmannin (0.1 and 1 μM), and FLA plus anti-mTLR5-IgG (1 μg/ml) (upper panel, n = 3). The graph depicts average intensity ratio of the bands compared to control (lower panel). Data are expressed as mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s post hoc test. **P < 0.01 vs. control; ^##P < 0.01 vs. FLA. d, e DMSO-containing DMEM served as control, while FLA was solved in DMSO-containing DMEM