Nitrocellulose membranes 0.45 μm

Total cellular proteins were extracted using RIPA lysis buffer containing phosphatase and protease inhibitors (Beyotime Biotechnology, China). The concentration of total protein was detected with a BCA Protein Assay kit (Beyotime Biotechnology, China). Equal amounts (20 μg) of protein were separated using 4–20% SDS-PAGE gels. The proteins were then transferred to nitrocellulose membranes (0.45 μm; Millipore, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies (Table 1) overnight at 4°C. After washing, the membranes were incubated with goat anti-rabbit secondary antibodies (Table 1). Finally, the membranes were visualized using the Odyssey CLx Infrared Imaging System (LI-COR Biosciences, Lincoln, Nebraska, USA). GAPDH and β-actin protein intensities were used as internal controls.

Cell pellets were homogenized in extraction buffer (50 mmol/L Tris-HCl, pH 6.8, 0.1% SDS, 150 μmol/L NaCl, 100 mg/L phenylmethylsulfonyl fluoride, 1 mg/L aprotinin, 1% NP-40 and 0.5% sodium orthovanadate), incubated at 4°C for 30 min, and centrifuged for 20 min at 12000 g/min. Total protein in the cell lysate was measured with use of the Bio-Rad colorimetric kit (Bio-Rad, Hercules, CA, USA). For western blot analysis, total protein was separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes (0.45 μm, Millipore, Billerica, MA, USA), which were incubated for 24 h at 4°C with the antibodies for α5-nAChR (1:500, ab41173 or ab166718), AKT(1:500, Epitomics Cat no:1085–1), P-AKT(1:500, Epitomics Cat no:2118–1), Caspase-3 (1:500, Epitomics Cat no:1087–1), Bcl-2 (1:500, Epitomics Cat no:1017–1), Survivin (1:500, Epitomics Cat no:2463–1) and GAPDH (1:1000; ab37168), then horseradish peroxidase-conjugated anti-mouse/rabbit IgG antibody (Santa Cruz Biotechnology) after a final wash. Signals were detected with use of an enhanced chemiluminescence kit (Amersham Pharmacia, Buckinghamshire, UK). GAPDH level was an internal standard.

The treated cells were harvested and lysed with RIPA lysis buffer (Beyotime, Shanghai, China) containing 1 mM PMSF (Beyotime, Shanghai, China), protease inhibitor cocktail, (Beyotime, Shanghai, China) and phosphatase inhibitors (Beyotime, Shanghai, China). Cytosol and nucleus proteins were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime, Shanghai, China). The concentrations of protein were determined by the BCA Protein Assay Kit (Thermo Scientific, LOT UA276918, USA). Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (0.45 μm) (Millipore, Ireland, LOT R1BB08547). The membranes were incubated with the following antibodies: β-catenin (1 : 1,000; CST 8480S), Lamin-b1 (1 : 1,000; CST 13435S), and GAPDH (1 : 1,000; CST 5174T). Subsequently, all membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (CST 7074 S) for 2 hr at room temperature. The bands were detected using the Chemiluminescence imaging system (BG-gdsAUTO710Pro, Shanghai) with Pierce^TM ECL Western Blotting Substrate (Thermo, #32106) and quantified using the Image J software (National Institutes of Health, USA).