Camkii

Western blot was performed according to the previous report 8 (link). Briefly, total proteins were separated by 10% SDS-PAGE gels and transferred to a PVDF membrane. After blocking with 5% skim milk, the PVDF membrane was incubated with primary antibodies and HRP-conjugated secondary antibodies. The primary antibodies are as follows: Wnt5a (1:1000, Abcam), p-ERK1/2 (1:1000, CST), ERK1/2 (1:1000, Protein Tech), p-CaMKII (1:1000, CST), CaMKII (1:1000, Protein Tech), α-Tubulin (1:5000, Protein Tech), GAPDH (1:5000, Protein Tech).

Total cellular proteins were extracted using RIPA lysis reagent (Aspen) containing a protease inhibitor cocktail (Aspen) with or without a phosphatase inhibitor (Bio-swamp). The protein concentration was measured by BCA protein assay reagent kit (Aspen) and mixed with 4 × SDS loading buffer (Bio-swamp) for denaturation in a 100 °C boiling water bath for 8 min. The proteins were then separated on 10% SDS-PAGE gels and transferred to PVDF membranes (Merck). The PVDF membranes were blocked with 5% skim milk for 90 min, washed three times, and then incubated with primary antibody at 4 °C overnight. Thereafter, the PVDF membranes were washed three times for 30 min and incubated with HRP-conjugated secondary antibodies for 90 min at room temperature. Finally, the membranes were again washed five times for 30 min, exposed to ECL developer (Aspen) and analyzed by Bio-Rad Image Lab software. The following primary antibodies used in this study were listed: Wnt5a (1:1000, Abcam), ERK1/2 (1:1000, Protein Tech), p-ERK1/2 (1:1000, CST), STAT3 (1:1000, Protein Tech), p-STAT3 (1:1000, CST), NF-κB p65 (1:1000, Protein Tech), p-NF-κB p65 (1:1000, CST), CaMKII (1:1000, Protein Tech), p-CaMKII (1:1000, CST), GAPDH (1:5000, Protein Tech), α-Tubulin (1:5000, Protein Tech).

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).