CYP2A and loading control analyses
Control lung membrane proteins (ranging from 25–150 μg) were separated by SDS–PAGE at constant voltages of 60V through a 4% acrylamide stacking gel and of 100V through a 10% acrylamide separating gel at room temperature (22°C). All immunoblotting procedures were carried out at room temperature unless specified otherwise. A portion of the gel (i.e. ~ 35 kDa to ~10 kDa) that did not contain our proteins of interest was stained with EZBlue Gel Coomassie Staining (Sigma; Oakville, ON, Canada) to quantify the protein loaded per well. Proteins in the remaining portion of the gel were transferred to nitrocellulose membrane at a constant current of 300 mAmp for 16 hours. Membranes were then rinsed three times for 5 minutes each in Tris-buffered saline (pH 7.4) with 0.1% Triton X-100 (TBST).
To detect CYP2A, membranes were blocked for 1 hour with a blocking solution consisting of 3% skim milk powder, 1% horse serum, and 0.5% bovine serum albumin (BSA) in TBST. Membranes were then incubated for 16 hours at 4°C with a polyclonal rabbit anti-CYP2A6 antibody (Sigma; Oakville, ON, Canada) diluted 1:1000 in 0.5% horse serum and 0.5% BSA in TBST. Membranes were rinsed 3 times for 5 minutes each in TBST, then incubated with fresh blocking solution for 1 hour, then with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (EMD Milipore; Etobicoke, ON, Canada) diluted 1:10000 in 0.5% horse serum and 0.5% BSA in TBST for 1 hour.
CYP2A proteins were visualized using an enhanced chemiluminescence (ECL) Immunoblot kit (ThermoFisher; Oakville, ON Canada). CYP2A proteins and Coomassie blue-stained proteins were imaged and quantified with a ChemiDoc™ MP imaging system equipped with ImageLab v6.0.1 build 34 imaging software. Exposure times of 30 and 60 seconds were used, enabling the detection of CYP2A and Coomassie blue-stained proteins without signal saturation.
After analysis for CYP2A, β-actin was quantified to further assess protein loading. Membranes were washed for 24 hours in TBST, incubated for 1 hour in fresh blocking solution, and then for 16 hours at 4°C with a mouse monoclonal anti-β-actin antibody (Sigma; Oakville, ON, Canada) diluted 1:1000 in 0.5% horse serum and 0.5% BSA in TBST. Membranes were then washed three times for 5 minutes each in TBST, re-blocked for 1 hour, then incubated for 1 hour in HRP-conjugated anti-mouse secondary antibody (EMD Millipore; Etobicoke, ON, Canada) diluted 1:10000 in 0.5% horse serum and 0.5% BSA in TBST. β-actin proteins on blots were imaged and quantified using the ChemiDoc™ MP imaging system described above using an automatic exposure time to minimize band saturation and maximize signal linearity.