Sc 28916

Tissues were homogenized using lysis buffer (Beyotime, China), and supernatants were collected after centrifugation at 12,000 × g for 15 min at 4°C. After quantitative analysis of protein concentration, total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Millipore, Billerica, Mass), blocked with 5% non-fat milk in Tris-buffered saline for 1 h at 37°C, and then incubated overnight at 4°C with primary antibodies. After incubation for 1 h at 37°C with secondary antibody (1:2,000; Beyotime), bands were seen using the enhanced chemilumine-scence kit (Beyotime) according to the manufacturer's protocol. The primary antibodies used were as follows: antineuregulin-1 (1:500, sc-28916, Santa Cruz Biotechnology Inc, Dallas, Tex), anti-γ-nAChR (1:500, sc-13998, Santa Cruz Biotechnology Inc, Dallas, Tex), anti-α7-nAChR (1:1,000, ab10096, Abcam, Ltd, Cambridge, Mass), anti-LC3II (1:500, 4108, Cell Signaling Technology, Danvers, Mass), anti-p62 (1:500, 5114, Cell Signaling Technology, Danvers, Mass), anti-GAPDH (1:1,000, AF1186, Beyotime Institute of Biotechnology, Jiangsu Province, China). All results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels.