FGF-2 exhibits neuroprotection via ERK MAPK signaling pathway. (A) Effects of MAPK and PI3K inhibitors on neuroprotection of FGF-2. U0126, MEK1/2 inhibitor U0126 (1 μM); SP, JNK inhibitor SP600125 (10 μM); SB, p38 inhibitor SB203580 (10 μM); WM, PI3K inhibitor wortmannin (2 μM). (B) Neuronal survival rate. Columns indicate mean with SEM from three independent experiments. ***: P < 0.001 compared with the cultures without inhibitors (one-way ANOVA with Dunnett’s post-hoc test). (C) Protein extracts from primary microglia were analyzed by immunoblotting with antibodies specific for phosphorylated and total ERK (ERK1/2). Cells were treated with 100 ng/ml FGF-2 for the indicated periods. *: P < 0.05 compared with untreated control (0 min) samples (one-way ANOVA with Dunnett’s post-hoc test). (D) Microglia were treated with U0126 (1 μM) or Wnt antagonist IWR-1-endo (300 nM) overnight and then 100 ng/ml FGF-2 for 15 min. Western blotting of phosphorylated and total ERK was performed. * indicates significant differences compared with untreated samples (*: P < 0.05, ***: P < 0.001); + indicates significant differences compared with FGF-2 treatment alone (+++: P < 0.001) by one-way ANOVA with Tukey’s post-hoc test. (E) Wnt promoter assay. HEK293T cells were transfected with Wnt promoter bearing the firefly luciferase reporter vector and Renilla luciferase reporter vector as a transfection control. After 24 h incubation, cells were treated with IWR-1-endo and U0126 overnight, and then treated with FGF-2 for 4 h. Cells were lysed and measured for luciferase activity. **: P < 0.01 compared with FGF-2 treatment alone (one-way ANOVA with Dunnett’s post-hoc test).