Molecular analyst image analysis software

For the analysis of COX-1, COX-2, and PLIN2 protein content, whole cell extracts were obtained by lysing the cell pellets with 80 mL of sample buffer (0.5 M Tris-HCl, pH 6.8, 20% SDS, 1% glycerol, 1 M β-mercaptoethanol, 0.1% bromophenol blue) and then boiled for 10 min at 100°C. Samples were resolved by electrophoresis (SDS-PAGE) on a 10% separation gel overlaid with a 5% stacking gel. After that, proteins were transferred to a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) using a Mini Trans-Blot^® (Bio-Rad Laboratories, Richmond, CA, USA), and the membranes were blocked for 1 h with 5% nonfat dry milk in TTBS (20 mM Tris, 100 mM NaCl and 0.5% Tween 20). Membranes were then incubated with a primary rabbit antibody against COX-1 or COX-2 or PLIN2 (Abcam, San Francisco, USA) overnight at 4°C or with β-actin (Sigma, San Louis, USA) for 1 h at room temperature. They were then washed and incubated with the appropriate horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1 : 1000 dilution, 1 h, at room temperature). Detection was by the enhanced chemiluminescence (ECL) method according to the manufacturer's instructions (GE Healthcare, Buckinghamshire, UK).
Band densities were quantified with a GS 800 Densitometer (Bio-Rad Laboratories, Richmond, CA) using Molecular Analyst® image analysis software (Bio-Rad Laboratories, Richmond, CA).

The protein expressions of HO-1 and Mfn1 of the left upper lung samples after 6 h were analyzed by Western blot. The proteins were extracted according to the instructions of the total protein and nuclear protein extraction kit (Thermo, USA), and the protein concentrations were detected using a BCA protein assay kit (Thermo, USA). Equal amounts of protein were fractionated using a 12% SDS-PAGE gel and were then transferred to a PVDF membrane (Millipore, USA). Blots were washed in triplicate for 5 min in TBS and were then incubated overnight at 4 °C with polyclonal rat antibodies against HO-1 (1:250, Abcam, UK) and Mfn1 (1:200, Santa, CA). Primary antibodies were diluted in a blocking solution containing 1% nonfat milk plus 0.5% BSA in TBS-0.05% Tween 20. After three washes with TBS-0.05% Tween 20, the blots were incubated at 37 °C for 1 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:3000 dilution, Beijing Kang-century Biotechnology Co, China). The blots were visualized with enhanced chemiluminesence (Bio-Rad, USA) according to the manufacturer’s instructions, and the relative densities of the bands were quantified by densitometry (Molecular Analyst Image-analysis Software, Bio-Rad, USA).