Western blotting

Samples were diluted to at least 1:5 with sample buffer, heated at 95 °C for 5 min, and then stored at 4 °C until they were used. The gel was run at 80 V for 10 min and then at 130 V for 3 h. Polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA) were soaked in methanol for 1 min and then placed in the “sandwich” chamber with 2 fiber pads and 2 filter papers, all soaking in old transfer buffer. The “sandwich” was transferred for 1.5 h at 100 V at 4 °C. The membranes were then shaken in 5% nonfat dry milk in PBS for 1 h on a shaker at room temperature; they were then incubated with a primary anti-KCNQ2 antibody (1:200) (Thermo Fisher, Waltham, MA, USA) in 1% milk at 4 °C on a shaker overnight. The next day, after it had been washed with PBST (phosphate buffer saline + Tween 20) 4 times for 10 min each time, the membrane was incubated with a secondary antibody (anti-rabbit) (1:3,000) (Gentex, Carbondale, PA, USA) in 1% milk prepared with PBS for ~ 1 h at room temperature, rinsed with PBST 4 times for 10 min each time, and then analysed using a western blotting detection kit (Advansta, Menlo Park, CA, USA). Anti-glyceraldehyde phosphate dehydrogenase (GAPDH) antibody was used as a loading control.