Mapping of the WNT-5A-induced signaling pathway leading to ERK1/2 phosphorylation in primary microglia. (A) Cells were treated with pharmacological inhibitors 10 minutes prior to WNT-5A challenge (300 ng/ml, 20 minutes). Immunoblotting analysis of P-ERK1/2 and P-MEK1/2 is shown. β-actin serves as loading control. Representative data from at least three experiments are shown and data are quantified in the bar graphs next to each inhibitor (error bars - s.e.m.; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) Lysates from microglia treated with WNT-5A in the absence or presence of D4479 were analyzed for the formation of PS-DVL3. (C) Table summarizes concentrations, targets and effects of the inhibitors used. Pharmacological inhibitors: D4476, CK1 inhibitor; M119, βγ inhibitor; U73122, PLC inhibitor; BIS,(bisindolmaleimide VIII), PKC inhibitor; wortmannin/LY294002, phosphatidylinositol-3'-kinase inhibitor; BAPTA-AM, Ca2+-chelator; SL327, MEK1/2 inhibitor. DVL3, disheveled 3; ERK1/2, extracellular signal-regulated kinase 1/2; MEK1/2, MAPK/ERK kinase 1/2; PKC, calcium-dependent protein kinase; PLC, phospholipase C; PS-DVL3, phosphorylated and shifted DVL3; s.e.m, standard error of the mean.