Anti bfgf

To confirm the difference in expressed proteins between the bEnd.3 cells and NSCs, proteins were extracted from ipsilesional brain tissue using a RIPA lysis buffer (Thermo Fisher, USA). For western blotting, equal amounts (30 μg) of protein extracts in a lysis buffer were used in a 12% SDS-PAGE analysis and transferred onto the polyvinylidene fluoride membranes. A 5% skimmed milk solution was used to block the membrane for 1 h at room temperature on a rocker. The housekeeping gene, β-actin, was employed as a loading control. Anti-Sox2 (1:1000, Santa Cruz, USA), anti-Nestin (1:1000, Abcam, UK), anti-VEGF (1:1000, Novus, USA), anti-bFGF (1:1000, Santa Cruz, USA), anti-CD31 (1:000, Abcam, UK), and anti-β-actin (1:1000, Santa Cruz, USA) antibodies were incubated with the membranes at 4°C overnight. After washing the membranes with TBST buffer, a horseradish peroxidase-conjugated anti-rabbit IgG antibody (Santa Cruz, USA) at a dilution of 1:20,000 or an anti-mouse IgG antibody (KPL, Inc., Gaithersburg, MD, USA) at a dilution of 1:10,000 was added to the corresponding primary antibodies, followed by incubation for 1 h at room temperature. Bands were detected using ECL reagent (Millipore, USA).

The cells were lysed in RIPA buffer and incubated for 10 min on ice and centrifuged at 12,000 rpm for 10 min at 4°C. Then, the whole protein samples were separated on 12% gradient SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA) that were blocked in 5% skim milk for 90 min and probed with the primary antibody anti-bFGF (1:500, sc-79, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. After washing three times with 0.1% Tween-20 in Tris-buffered saline (TBS), the membrane was incubated with the secondary antibody for 1 h at room temperature. Consequently, the target protein was detected using a ChemiDoc^TM XRS+ Imaging System (Bio-Rad).

All reagents we used were commercially available. Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Invitrogen (Carlsbad, California). Recombinant human bFGF was purchased from Sigma (Sigma‐Aldrich, St. Louis, Missouri). Anti‐GFAP, anti‐bFGF, anti‐p62, anti‐NeuN, and anti‐GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California). Anti‐GAP43, anti‐LC3, anti‐Beclin‐1, and anti‐Nestin antibodies were purchased from Abcam (CB, United Kingdom). Goat anti‐rabbit and anti‐mouse IgG‐HRP, goat anti‐chicken IgY H&L, donkey anti‐goat IgG H&L were purchased from Santa Cruz Biotechnology. An enhanced chemiluminescence kit and CM‐DiI were purchased from Bio‐Rad (Hercules, California). Thapsigargin (TG) and 3‐methyladenine (3‐MA) were purchased from Sigma‐Aldrich. The autophagy activator rapamycin (RAPA) was purchased from Cell Signaling Technology. All other reagents were purchased from Beyotime Institute of Biotechnology (Shanghai, China) unless otherwise specified.