β actin

Cell lysates were prepared using radioimmunoprecipitation assay lysis buffer (MCE, HY-K1001). Equal amounts of lysates (15 μg) were separated using 4 to 20% polyacrylamide electrophoresis gel and transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA). After being blocked with 5% skim milk, the membranes were incubated with primary antibodies at 4°C overnight and secondary antibodies for 1 hour at 37°C. Signals detected using the ECL kit (K-12094-D50, Advansta, CA, USA) were quantified using ImageJ software and normalized to β-actin levels. The following primary antibodies were used: Pan-Kla (diluted 1:1000; PTM-1401RM), Ikzf1-K164la (PTM), Ikzf1 (diluted 1:1000; 14859, CST), β-actin (diluted 1:3000; Proteintech, 20536-1-AP), and FLAG (diluted 1:1000; 14793, CST).

Protein was extracted from preconditioned cAT-MSC-derived exosomes and DH82 using the Pro-Prep protein extraction solution (Intron Biotechnology). Concentration of the protein samples was analyzed using the DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Nuclear proteins were isolated using the Cell Fractionation Kit-Standard (Abcam, Cambridge, MA, USA). For western blot assays, 25 μg of proteins were loaded and separated by SDS-PAGE. Bands from SDS-PAGE were transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), which were then blocked with 5% non-fat dry milk and Tris-buffered saline. Membranes were incubated with primary antibodies against HIF-1α (1:500; LifeSpan BioSciences, Seattle, WA, USA), COX-2 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), STAT3 (1:500, LifeSpan BioSciences), phosphorylated (Tyr705) STAT3 (1:500, LifeSpan BioSciences), lamin A (1:500, Santa Cruz Biotechnology) and β-actin (1:1000, Santa Cruz Biotechnology) at 4°C overnight. The membranes were subsequently incubated with the appropriate secondary antibody for 1 h. Using an enhanced chemiluminescence detection kit (Advansta, Menlo Park, CA, USA), immunoreactive bands were detected and normalized to the housekeeping protein (β-actin).

Western blot was used to detect the expression of BDNF, 5-hydroxytryptamine (5-HT), SHH, GLI, patched (PTCH), and smoothened (SMO) in the hippocampus. Total protein was extracted from tissues using RIPA lysis buffer. BCA protein assay kits were used for protein quantification. SDS-PAGE loading buffer was mixed, the protein was adsorbed on PVDF membrane by gel electrophoresis, sealed with 5% skim milk, and incubated with primary antibodies BDNF (28205-1-AP, 1: 1000, proteintech), 5-HT (ab85615, 1 μg/mL, Abcam), SHH (20697-1-AP, 1: 1500, proteintech), GLI (66905-1-Ig, 1: 3000, proteintech), PTCH (#2468, 1: 1000, proteintech), SMO (66851-1-Ig, 1: 4000, proteintech), and β-actin (66009-1-Ig, 1: 5000, proteintech). Afterward, HRP secondary antibody was incubated. ECL chemiluminescence solution (K-12045-D50, Advansta, USA) was used for chromogenic exposure with β-actin as an internal reference to detect expression levels.