Western blotting was performed as described previously (Bellanger et al, 2011 (link)). Rabbit anti-BDNF, anti-NGF, anti-NT3, anti-p75NTR, goat anti-sortilin, and mouse anti-PARP-1 antibodies were purchased from Santa Cruz Biotechnology. Mouse anti-TrkB, anti-TrkA, and anti-TrkC antibodies were purchased from R&D Systems. Anti-phospho TrkB (pY817) and anti-phospho-TrkC (Y820) were purchased from Abcam (Epitomics, Burlingame, CA, USA) and anti-phospho-TrkA (Y490) from Cell Signalling. Protein-loading control was performed with anti-βactin Ab (Sigma). Visualisation of immunocomplexes was accomplished using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Darmstadt, Germany). Western blots were scanned using a bioimaging system (Genesnap; Syngene Europe, Cambridge, UK).
Co-immunoprecipitations were performed in accordance with the manufacturer's instructions (Catch and Release kit, Millipore) with 500 μg of proteins and anti-p75NTR for 1 h at room temperature. Finally immunoprecipitates were subjected to SDS–polyacrylamide gel electrophoresis, before analysis by western blotting (with anti-Trk, anti-sortilin and anti-p75NTR).
Co-immunoprecipitations were performed in accordance with the manufacturer's instructions (Catch and Release kit, Millipore) with 500 μg of proteins and anti-p75NTR for 1 h at room temperature. Finally immunoprecipitates were subjected to SDS–polyacrylamide gel electrophoresis, before analysis by western blotting (with anti-Trk, anti-sortilin and anti-p75NTR).
