Ar1183

Fresh lumbar spinal cord tissue was extracted and put into RIPA lysate (AR0102-100, Boster), adding protease inhibitor PMSF(AR1178, Boster) and phosphatase inhibitor (AR1183, Boster). BCA protein concentration assay kit (AR0146, Boster) was used to determine the protein concentration.
The total proteins from the spinal cord were separated by electrophoresis using 10% SDS–polyacrylamide gel, then transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h at room temperature, then incubated overnight at 4 °C with the following antibodies: Rabbit Anti-SHH Polyclonal Antibody (bs-1544R, Bioss), Rabbit Anti-Gli-1 Polyclonal Antibody (bs-1206R, Bioss), Phospho-AKT (Ser473, Cell Signaling Technology), AKT polyclonal antibody (AP0095, Bioworld) or β-actin (AP0060, Bioworld) antibodies. After washing, the membranes were incubated with Goat anti-Rabbit IgG (BA1054, Boster) for 1 h at room temperature. The blots were visualized by the chemiluminescence-based detection kit (ECL Kit, Boster). Densitometry analysis was performed with a System Gel Doc XR + IMAGE LAB (Bio-Rad, USA) and quantified with NIH Image software (Image J).

Total protein was extracted using RIPA buffer (AR0102, Boster) supplemented with a protease inhibitor cocktail (HY-K0010, MCE) and phosphatase inhibitor (AR1183, Boster). The concentration of extracted proteins was measured using a BCA Protein Assay Kit (PC0020, Solarbio), and equal amounts of extracted proteins were loaded onto SDS-PAGE. The size-separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore) for blotting. After blocking with 5% BSA blocking buffer (SW3015, Solarbio), membranes were incubated overnight at 4 °C with the following specific primary antibodies: anti-YAP antibody (13584-1-AP, Proteintech), anti-phosphorylated YAP antibody (Ser127) (13008 T, Cell Signaling Technology), anti-NANOG antibody (sc-293121, Santa Cruz), anti-OCT4 antibody (sc-5279, Santa Cruz), and anti-GAPDH antibody (10494-1-AP, Proteintech). Following washing, membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (SA00001-2, Proteintech). Protein expression was detected using ECL western blotting substrate (PE0010, Solarbio), and the membranes were imaged using a membrane imaging system (Clinx, ChemiScope S6).

Hippocampal tissues were prepared as described above. Cells (see below) were washed twice with ice-cold PBS. The tissues and cells were homogenized in cold RIPA buffer (AR0102, BOSTER) containing a cocktail of PMSF (AR1179, BOSTER) and protein phosphatase inhibitor (AR1183, BOSTER). The homogenates were then centrifuged (16,000×g, 30 min, 4 °C), and the supernatants were collected. The total protein concentrations of the samples were measured by a BCA Protein Assay Kit (PC0020, Solarbio). Total protein for each sample was diluted 1:1 with loading buffer (AR0131, BOSTER) and boiled for 5 min at 95 °C. Equal amounts of total protein were separated by 12% or 15% SDS-PAGE (AR0138, BOSTER) and transferred to PVDF membranes (0.45 μm or 0.22 μm, Millipore). Following blocking with 5% BSA for 2 h at room temperature, the membranes were incubated with desired primary antibodies overnight at 4 °C, and then HRP-conjugated secondary antibody for another 2 h at room temperature. After several washes, the protein bands were developed with ECL Western Blot Detection kit (P0018FS, Beyotime) and detected using Azure c300 Chemiluminescent Western Blot Imaging System (Azure Biosystems). The band intensity was analyzed with AlphaView SA (Fluorchem FC3, ALPHA).