Lc3 and cleaved caspase 3

Penicillin-streptomycin solution, trypsin-ethylene diamine tetra acetic acid (EDTA) solution, Neurobasal Medium (NBM), B27 supplement, Glutamax and fetal bovine serum (FBS) were obtained from Life Technologies/Gibco (Carlsbad, CA, USA). Minimum Essential Medium (MEM) and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from GE Healthcare/HyClone (Logan, UT, USA). Methyllycaconitine citrate salt (MLA) and PNU-282987 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The immunoblotting and immunocytochemistry antibodies targeted LC3 and cleaved-caspase-3 (Cell Signaling Technology, Danvers, MA, USA), α7nAchR (Abcam Inc., Cambridge, MA, USA), p62 (Millipore, Temecula, CA, USA) and β-actin (Sigma-Aldrich).

Cells were plated at density of 4.8×10⁴ cells per well in 6-well plates and treated with vehicle or drugs. After the indicated time of treatment, cells were lysed by brief sonication in whole-cell lysis buffer (62.5 mM TRIS, 10% glycerol, 1% SDS, pH 6.8), and cellular proteins were collected in the supernatant fraction after centrifugation at 14,000 g for 10 minutes. Proteins were reduced by heating at 100°C for 5 minutes in 50 mmol/L DTT with 0.05 bromphenol blue, electrophoresed in 4–20% Tris-HCl gels, and transferred onto nitrocellulose using the NuPage electrophoresis system (Invitrogen, Carlsbad, CA). The membranes were probed with antibodies to LC3 and cleaved caspase 3 (Cell Signaling, Danvers, MA USA) and actin (Santa Cruz Biotechnology Company, Santa Cruz, CA). Membranes were treated with appropriate secondary antibody, exposed to enhanced chemiluminescence solutions (Invitrogen, Carlsbad, CA) and visualized using the Molecular Imager Gel Documentation System.