Figure 3

WNT-5A-induced ERK1/2 phosphorylation requires activation of Gα _i/o . (A) Primary microglia, pretreated with PTX (100 ng/ml, overnight), were stimulated with 300 ng/ml WNT-5A for 30 minutes and their total cell lysate was analyzed for P-ERK1/2 by immunoblotting. β-actin serves as a loading control. Densitometric analysis of three independent experiments is summarized in the bar graph (error bars - s.e.m.). **, P < 0.01. (B) In a similar stimulation paradigm, cells were lysed after two hours and the lysate was analyzed for the formation of PS-DVL3 (filled triangle: DVL3, open triangle: PS-DVL3). n = 3. (C) Indirect immunocytochemistry and confocal microscopy were used to detect and localize P-ERK1/2 in ctrl, WNT-5A (300 ng/ml, 30 minutes) and WNT-5A/pertussis toxin (PTX, 100 ng/ml)-treated mouse primary microglia. IBA1 was used as a microglia marker and DAPI as nuclear counterstain. Arrows mark WNT-5A-responsive microglia with increased P-ERK1/2 levels (n = 3). Size bar = 50 μm. ctrl, control; DAPI, 4',6-diamidino-2-phenylindole; DVL, disheveled; ERK1/2, extracellular signal-regulated kinase 1/2; IBA1, ionized calcium-binding adaptor protein1; n, number; P-ERK1/2, ERK1/2 phosphorylation; PS-DVL, phosphorylated and shifted DVL; s.e.m., standard error of the mean.