Bdnf b 250

A list of oligonucleotides and antibodies used for the study are listed in Supplementary Tables 2, 3, respectively. K252a (1863), thapsigargin (1138), U73122 (1268/10) and U73343 (4133/10) were from Tocris. Fluo-4-AM (F14201), Powerload (P10020) and Probenecid (P36400) were from Thermo Fisher Scientific. EGTA (E3889) was from Sigma-Aldrich. Brain-derived neurotrophic factor (BDNF) (B-250) was from Alomone labs.

For phospho-Trk and phospho-Erk activation, CGN and PC12 cells were incubated in serum-free DME supplemented with 0.1% bovine serum albumin (BSA) and 2mM L-glutamine and 100μg/ml penicillin and 100μg/ml streptomycin (DMEB) for 1h prior to treatment with BDNF, proBDNF and HBpF-proBDNF. The anti-biotin antibody was from Jackson Immunoresearch, anti-Flag and anti-beta III-tubulin were from Sigma, anti-BDNF (N-20) was from Santa-Cruz Biotechnology, Inc. and anti phospho-TrkA (Tyr490) was from Cell Signaling Technology. Antibodies against p75NTR, total TrkA and total TrkB have been previously described [20 (link)–23 (link)]. Anti-Sortilin and anti-SorCS2 were gifts from Dr. Barbara Hempstead, Rhodamine-tagged phalloidin was from Life technologies and Hoechst 33342 was obtained from Molecular Probes. Horseradish-peroxidase-conjugated and fluorescent dye-conjugated secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). BDNF (B250) and proBDNF (B240) was purchased from Alomone labs (Jerusalem, Israel). Cy3-Streptavidin was purchased from Invitrogen and GM6001 was purchased from Calbiochem.