Chen LY, Rex CS, Sanaiha Y, Lynch G, Gall CM. Learning induces neurotrophin signaling at hippocampal synapses. Proc Natl Acad Sci U S A. 2010 Apr 13;107(15):7030-5. PubMed Abstract

REAGENTS/MATERIAL:
Studies of the effects of synaptic activity on BDNF signaling employed three antisera directed against tyrosine residues that are phosphorylated (p) with Trk activation (TrkY490, TrkBY515, and TrkBY816). The TrkBY515 and TrkY490 residues mediate SHC binding, whereas TrkBY816, which lies within the C terminus of TrkB only, binds phospholipase C-gamma. Immunoprecipitation confirmed that the anti-TrkBY515 does not bind detectable levels of TrkA in adult rat hippocampus.
Immunofluorescence: Sections to be compared were processed simultaneously for double immunofluorescence using mixtures containing anti-mouse and anti-rabbit primary antibodies. Antibodies used included mouse monoclonal anti-PSD95 (6G6-1C9) (Affinity Bioreagents), rabbit anti-pTrkY490, recognizing a site conserved across Trks A, B, and C (Cell Signaling), rabbit anti-pTrkBY515 (Abcam), anti-pTrkBY816 (#18664–110207-4; a gift from M. Chao, New York University, New York, NY), mouse monoclonal anti-TrkB (47/TrkB) (BD Biosciences), rabbit anti-pan Src (Biosource), rabbit anti-Src (Abcam) and rabbit anti-BDNF (Biosensis).
Western Blot Analysis: Samples were processed for Western blot analysis using 8% SDS polyacrylamide gel electrophoresis; antibodies include rabbit antisera to pTrkBY816 (see above), rabbit anti-TrkA (Millipore) and rabbit anti-TrkB (Millipore).