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. 2011 Aug 31;31(35):12426–12436. doi: 10.1523/JNEUROSCI.0685-11.2011

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Copyright © 2011 the authors 0270-6474/11/3112426-11$15.00/0

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Figure 6. — Kinase-active TrkB activates Vav2 independently of the ERK/PI3K- and PLCγ-interacting sites. A, Coexpression of TrkB and Vav2 increases phospho-Vav2 (Y172) in HEK293T cells. HEK293T cells were transfected with T7-tagged wild-type or ΔSH2 and Flag-tagged wild-type or mutant TrkB. Total cell lysates were blotted with anti-phospho-Vav2 (Y172), anti-T7 (Vav2), anti-phospho-TrkB (Y490), and anti-TrkB antibodies. A–E, Results are representative findings from three independent experiments. B, TrkB binds Vav2 and Vav3 in HEK293T cells. HEK293T cells were transfected with HA-tagged Vav2 or Vav3 and Flag-tagged TrkB. Total cell lysates were immunoprecipitated with anti-Flag antibody, then immunoblotted with anti-HA (Vav) and anti-Flag (TrkB) antibodies. Whole-cell lysates were blotted with anti-HA (Vav) antibody. C, TrkB binds Vav2 independently of TrkB kinase activity. T7-tagged Vav2 was coexpressed with Flag-tagged TrkB (wild-type), TrkB-KD (K571N), TrkB-Y490F, or TrkB-Y785F. Total cell lysates (TCLs) were immunoprecipitated with anti-Flag antibody, then immunoblotted with anti-Vav2 and anti-Flag (TrkB) antibodies. Whole-cell lysates were blotted with anti-Vav2 antibody. D, Deletion analysis of Vav2 identifies the DH/PH/ZF region as sufficient to mediate coassociation with TrkB. Domain composition of full-length and Vav2 deletion mutants that were generated. C, Calponin homology domain; A, acidic domain; DH, Dbl homology domain; P, pleckstrin homology domain; Z, zinc finger domain; P, proline-rich domain; 3, Src homology type 3 domain (SH3); 2, Src homology type 2 domain (SH2). HEK293T cells were transfected with Flag-tagged TrkB (wild-type) and T7-tagged Vav2 (wild-type), Vav2-ΛCH/AD, Vav2-Λadaptor, or Vav2-DH/PH/ZF. Total cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-T7 (Vav2) and anti-Flag (TrkB) antibodies. Whole-cell lysate was blotted with anti-T7 (Vav2) antibody. E, TrkB's kinase domain is required for its interaction with Vav2. T7-tagged Vav2 was coexpressed with wild-type or Δ kinase domain (ΔKD)-TrkB. Total cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Vav2 and anti-Flag (TrkB) antibodies. Whole-cell lysate was blotted with anti-Vav2 antibody.