Figure 1.

BDNF stimulates the transient activation of Vav2 in neurons. A, Vav2 antibodies are highly specific. Western blot of whole hippocampi from P15 WT or Vav2^−/−3^−/− (DKO) mice with anti-Vav2 antibody (Cowan et al., 2005). B, E18 rat cortical neurons were cultured for 6 d and then stimulated with BDNF (100 ng/ml) over a time course. Vav2 was immunoprecipitated and blotted with antiphosphotyrosine antibody (4G10). Total cell lysates (TCLs) were blotted with anti-phospho-TrkB (Y490) and anti-TrkB antibodies. The results are a representative finding of five independent experiments. C, Coexpression of Vav2 or Vav3 and TrkB increases phospho-Vav in HEK293T cells. HEK293T cells were transfected with T7-tagged Vav2 or HA-tagged Vav3 and Flag-tagged TrkB. To examine Vav2 Y172 phosphorylation, whole-cell lysates were blotted with anti-phospho-Vav2 (Y172) antibody. To examine Vav3 phosphorylation, total cell lysates were immunoprecipitated with anti-HA antibody, then immunoblotted with antiphosphotyrosine (4G10) and anti-HA (Vav) antibodies. Total cell lysates were blotted with anti-pTrkB (Y490) antibody. The results are a representative finding of three independent experiments. D, BDNF stimulates activation of Vav2 independently of Src kinase activity. E18 cortical neurons were cultured for 6 d and then incubated with DMSO, PP2 (5 μm), or PP3 (5 μm) for 30 min before stimulation with BDNF (100 ng/ml) over a time course. Vav2 was immunoprecipitated and blotted with antiphosphotyrosine antibody (4G10). Total cell lysates were blotted with anti-phospho-TrkB (Y490), anti-TrkB, anti-phospho-ERK1/2 (T202/Y204), anti-ERK1/2, anti-phospho-Src (Y418), and anti-Src antibodies. The results are a representative finding of two independent experiments.