Recombinant bdnf protein

Immunoblot was performed to confirm the intracellular signaling pathway through the BDNF–TrkB pathway. To induce differentiation of SH-SY5Y cells (Korea Cell Line Bank), 10 μM of retinoic acid (Sigma-Aldrich, Saint Louis, MO, USA) was used. Recombinant BDNF protein (Peprotech, Rocky Hill, NJ, USA) was treated as positive control and supernatant of BDNF-eMSCs, together or alone with recombinant human TrkB-hyFc (Sino Biological, Beijing, China), were incubated with differentiated SH-SY5Y cells for 1 h at the 37 °C, 5% CO₂ incubator. BDNF-eMSCs were harvested and lysed with RIPA buffer (Thermo Fisher Scientific). Lysate proteins were distinguished using SDS-PAGE, transferred to nitrocellulose membranes (Thermo Fisher Scientific), and incubated with blocking buffer (Thermo Fisher Scientific) for 30 minutes. Immunoblots were performed using primary antibodies against TrkB, PLC-γ, AKT, ERK, Phospho-TrkB (Tyr706/Tyr707), Phospho-PLC-γ (Tyr783), Phospho-AKT (Ser473), Phospho-ERK (Thr202/Tyr204) (Cell Signaling, Danvers, MA, USA), and ACTIN (MP Biomedicals, Santa Ana, CA, USA). Horseradish peroxidase (HRP)-conjugated mouse (GeneTex, Irvine, CA, USA) and rabbit (Cell signaling) secondary antibodies were detected with ECL reagent (Thermo Fisher Scientific) and a ChemiDoc XRS+ System (Bio-Rad, Hercules, CA, USA).