2.6 Immunoblotting

Proteins were extracted from ReNcell VM using RIPA lysis buffer (50 mM Tris, 1% NP40, 0.1% SDS, 0.15 M NaCl, 0.5% Na deoxycholate) supplemented with protease inhibitor (Roche #04693124001) and phosphatase inhibitor (Sigma#P5726). For phospho-ERK analysis, ReNcell were treated with different concentrations of human BDNF for 5 min (PHC7074, Life Technologies).

Protein concentration was estimated by measuring absorbance at 570 nm (Spectramax, Molecular Devices) following reaction with the BCA Protein Assay Kit Pierce™ (Thermo Fisher # 23227) and quantified by comparison of a standard curve of bovine serum albumin (Sigma # A3912). Fourty μg amount of protein extract was diluted in RIPA buffer, complemented with sample buffer 5X (Tris-base pH 6.8 312 mM, Glycerol 50%, SDS 10%, 2-mercaptoéthanol 25%, bromophenol blue 0.01%), heated 5 min at 100°C. Samples were separated in Bolt 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel (Thermo Fisher # NW04125BOX), run at 1 h 100V in Bolt MOPS SDS Running Buffer (Thermo Fisher # B0001), transfer on PVDF 0.45microM Immobilon P membrane (Millipore # IPVH00010) in transfer buffer (Tris-base 25 mM, Glycine 192 mM, methanol 10%, SDS 0.005%) 90 min at 80 V. Membranes were colored with Fast green 0.01% (in methanol 20% and acetic acid 5%). Membranes were blocked in TBST buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.6, Tween 0.05%) with BSA (Sigma # 3912) 5%. Membranes were incubated with primary antibodies P-ERK Phospho-p44/42 MAPK (Erk1) (Tyr204)/(Erk2) (Tyr187) (D1H6G) Mouse mAb (Cell Signaling #5726), ERK p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb (Cell Signaling #4695) and recombinant monoclonal mouse antibody anti-TrkB AG424 (Roussel-Gervais et al., 2020) and in secondary antibodies Goat Anti-Mouse IgG (H + L)-HRP Conjugate (Bio-Rad #1706516) and Goat Anti-Rabbit IgG (H + L)-HRP Conjugate (Bio-Rad #1706515). Peroxidase-dependent bioluminescence was revealed with ECL Western Blotting (Witec # K-12042-D10), using Fusion Solo (Witec AG).