Neuron , Volume 63 Supplemental Data CCM 2 Mediates Death Signaling by the TrkA Receptor Tyrosine Kinase

Cells, antibodies and DNA constructs: Medulloblastoma D283MED-TrkA3 (MBTrkA) cells were kindly provided by Virginia Lee. cDNA clones and constructs were as previously described (Bronfman et al., 2003; Tcherpakov et al., 2002). TrkA mutations are numbered relative to the Shc site tyrosine 490. Trk kinase dead mutants and deletions were kindly provided by Moses Chao (Yano et al., 2000; Yano et al., 2001). TrkAΔ2 (TrkA with juxtamembrane deletion of residues 473-493) and TrkB(AJM), a TrkB chimera containing a TrkA juxtamembrane region were previously described (Chen et al., 2005). Primary antibodies and dilutions were as follows: antiTrkA RTA, a kind gift of Dr. Reichardt (1:1000 for WB); pan trk (C14, 1:200, Santa Cruz), anti phopho-tyrosine (PY99, 1:200, Santa Cruz), anti β-tubulin (H235) (1:200 Santa Cruz); anti-HA polyclonal (1:200 for i.p., Santa Cruz); anti HA mouse monoclonal (16B12, 1:1000 for WB, Covance); anti-HA agarose-conjugated (Sigma); FLAG M2 monoclonal (Sigma, 1:1000 for WB), anti-FLAG M2 affinity gel and FLAG peptide (Sigma); anti TrkA goat antibody (10 μg/ml for immunocytochemistry, R&D); Dapi (1:1000 for immunocytochemistry); anti His (Sigma, 1:1000 for WB); p75 (1 μg/ml Upstate); streptavidin dynbeads (Dynal). Polyclonal rabbit antisera were raised against the murine CCM2 PTB (residues 1-227) and used at 1:1000 for immunocytochemistry. Secondaries included Cy2-conjugated donkey anti-mouse (1:1000) or Cy5-conjugated donkey anti goat (1:500) (Jackson Immuno Research).