Western Blot Analysis of ELF-EMF Effects

Western blot analysis was performed as described by M He, et al [29 (link)]. After ELF-EMF exposure, protein samples were harvested and lysed in RIPA buffer (Thermo Scientific, USA) containing a cocktail of protease inhibitors (Roche, USA). The protein concentration was determined using a BCA protein assay kit (Takara, Japan). Equal amounts of protein were load for SDS-PAGE. After electrophoresis, the proteins were transferred onto a polyvinylidene fluoride membrane (Bio-Rad, USA). Then, the membranes were blocked and incubated with various primary antibodies at 4°C overnight. Mouse anti-mouse TRPC1 (1:1000, Santa Cruz, USA), rabbit anti-mouse NeuroD (1:500, Santa Cruz, USA), goat anti-mouse Ngn1 (1:500, Santa Cruz, USA), and mouse anti-mouse GAPDH (1:5000, Abcam, USA) were used as primary antibodies. The next day, the membranes were washed in Tris-buffered saline-Tween 20 to remove unbound primary antibodies, incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:1000, Beyotime, China), and finally examined using an electrochemiluminescence system (Thermo Fisher Scientific, USA). The bands were imaged and analyzed using a ChemiDoc XRS + System with Image Lab Software (Bio-Rad, USA).