Horseradish peroxidase conjugated anti mouse rabbit igg antibody

Cell monolayers were washed twice with PBS, harvested, and lysed with ice-cold assay buffer (Sigma-Aldrich) after which protein lysates (20 μg) were subjected to SDS-PAGE and Western blotting. The antibodies used for immunoblotting included those raised against cyclin D1, cyclin E2, cyclin D3 (1:500; Cell Signaling Technology), GAPDH (1:1000; Abcam) and horseradish peroxidase-conjugated anti-mouse/rabbit IgG antibody (1:10000; Santa Cruz Biotechnology). After a final wash, signals were detected with use of an enhanced chemiluminescence kit (Amersham Pharmacia, Buckinghamshire, United Kingdom). GAPDH levels were used as an internal standard.

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.