Ecl kit reagents

Total proteins were extracted from the NSCs (1 × 10⁶ cells) on different substrates (TCP, Si, or Au nanostrip array) with or without a rotating magnetic field (300 rpm) at 5 days using RIPA lysis buffer (Beyotime Biotechnology, China). The BCA Protein Assay Kit (Beyotime Biotechnology) was used to quantify total protein concentrations. After dilution with 5× loading buffer, equal amounts of protein were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were electrophoresed initially at 110 V for 90 min or longer, and then transferred to polyvinylidene difluoride membranes at 300 mA for 1 h or longer using a wet transfer system. The membranes were blocked with 5% nonfat milk for 1 h at room temperature. Next, the membranes were incubated with primary antibodies against GAPDH (1:2000, Affinity, China), Tuj1 (1:2000, Abcam, UK), MAP2 (1:2000, Abcam), ChAT (1:2000, Abcam), GAD65 (1:2000, Proteintech, USA), c‐Fos (1:2000, Proteintech), CaMKII (1:2000, Proteintech), and p‐CaMKII (1:1000, Affinity) overnight at 4 °C. The membranes were washed with tris‐buffered saline containing with 0.1% Tween 20 detergent and then incubated with secondary antibodies at room temperature for 1 h. Finally, the chemiluminescent signal was developed using ECL kit reagents (Millipore, USA) and detected using X‐OMAT BT Film (Kodak, USA).

Tissues were homogenized with RIPA containing PMSF and protease/phosphatase inhibitors following centrifugation at 4°C at 13,800 × g for 10 min. Then, 5× loading buffer was added to the protein supernatant and total protein was quantified using a BCA assay kit CWBIO (Haimen, Jiangsu, China). Equal amounts of protein were separated by SDS-PAGE and then transferred to PVDF membranes. After blocking in 5% nonfat milk for 2 h, blots were probed using the following primary antibodies: Shh, Gli-1, Patch, p-CREB, CREB, BDNF, p-Akt, Akt, NF-κB, p-NF-κB, and β-actin at 4°C overnight. Secondary antibodies were then incubated with the membranes at 37°C for 60 min. The chemiluminescent signal was developed with use of ECL kit reagents (MILLIPORE, USA) and then detected with the use of the Tanon Imaging System (Tanon-4600). Densities of protein bands were semiquantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).