2.6. Western Blot

Fresh frozen mice lung tissues were sonicated and homogenized in ice using the Radio-Immunoprecipitation Assay (RIPA) buffer composed of 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 1 mM sodium orthovanadate, 1 mM NaF, and a protease inhibitor tablet. The samples were analyzed for protein content using a Bradford protein assay (BioRad) according to manufacturer's instructions. Each sample was quantified, and then 9 μg of protein was loaded onto a 12% Tris-glycine SDS-polyacrylamide gel, according to the manufacturer's protocol (BioRad). The gel was transferred to a PVDF membrane by electrophoresis at 30 V for 16 h. The membrane was blocked in PBS with 0.1% Tween 20 (PBS-T) and 10% nonfat milk at room temperature for 2 h. All antibodies were diluted in the same buffer (PBS-T) with 1% nonfat milk.

The membrane was then probed with rabbit polyclonal HO-1 antibody (Abcam, ab13243, 1 : 20,000) and then incubated for 1 h at room temperature. After incubation, the membrane was washed five times with PBS-T and incubated with horseradish peroxidase–conjugated goat anti-rabbit IgG (Millipore, ap307p, 1 : 20,000) for 1 h at room temperature. After washing five times with PBS-T, an ECL system (Clarity Western ECL Blotting Substrate, Biorad) was used for detection of the proteins in a ChemiDoc XRS+ System (Biorad). The HO-1 expression was calculated by densitometry, in the software ImageJ (version 1.50b) of the HO-1 bands in the immunoblot relative to the housekeeping protein β-actin (rabbit monoclonal to β-actin, Novus biologicals, NB600-501, 1 : 500,000).