Quantiscan software

In this study, the differentially expression proteins PARK7 and FABP5 after day 14 of smoke inhalation were selected for western blot (WB) analysis on days 1, 2, 7 and 14. Protein extracts were prepared by homogenization with protein lysates. Protein concentrations were quantified with an Enhanced BCA Protein Assay Kit (Thermo Fisher Scientific, Rochester, N.Y., USA). Protein samples were run on a 12% sodium dodecyl sulfate polyacrylamide gel for electrophoresis and were transferred to a polyvinylidene fluoride membrane. The membrane was blocked with non-fat dried milk for 1 h and was incubated with the following primary antibodies: rabbit anti-human FABP5 (1:1000 dilution), rabbit anti-human PARK7 (1:500 dilution) and mouse monoclonal IgG1 to GAPDH (1:3000 dilution). Primary antibody binding was detected using horseradish peroxidase-conjugated secondary antibody (1:10000 dilution). Detection was achieved with the ECL chemiluminescence kit (Pierce). Bands from three separate western blots were analysed by Quantiscan software (Biosoft, Great Shelford, Cambridge, UK).

Complementary to coronary flow measurement, we assessed the expression of eNOS, SIRT1, Akt, and P-Akt proteins in freeze-clamped hearts. A piece of left ventricle tissue (≈60 mg) was homogenized in a RIPA lysis buffer and centrifuged at 14,000 rpm for 15 min at 4 °C. Total protein concentration in the supernatant was determined using the Pierce BCA protein assay kit. Equal amounts of proteins (90 µg for eNOS and SIRT1, 50 µg for Akt and P-Akt) were separated by 8% or 10% polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After blocking with 5% skim milk, membranes were incubated overnight at 4 °C with eNOS (1/1000), SIRT1 (1/1000), Akt (1/1000), P-Akt (Ser 473) (1/500), or Actin (1/2000) primary antibodies. Second, membranes were incubated with HRP-conjugated antibodies (1/2000). The immunoblots were developed and the protein signal was quantified using the Quantiscan software (Biosoft, Cambridge, U.K.). The intensity of each protein signal was normalized to the corresponding β-actin stain signal. Data are expressed as ratios between the protein and the corresponding β-actin signal density, except for P-Akt, which was expressed according to Akt.

Cells were lysed in Nonidet P-40 (NP40) lysis buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol and 1% NP40]. Equal amounts of total protein, as determined by the BCA protein assay kit (Pierce, Rockford, IL, USA), were subjected to SDS-PAGE on 8% polyacrylamide gels and transferred to Hybond ECL nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). Blots were stained with Ponceau S to ensure protein amounts were equal. For immunodetection, blots were soaked in blocking buffer [1% Blocking Reagent (Roche, Mannheim, Germany) in 0.05% Tween 20-PBS] for 1 h and incubated with primary antibody in blocking buffer overnight at 4 °C. Blots were then washed in 0.05% Tween 20-PBS and incubated with either goat anti-mouse IgG (1:20,000, GE Healthcare, Little Chalfont, UK) or goat anti-rabbit IgG (1:20,000, GE Healthcare) peroxidase-labeled antibodies in blocking buffer for 1 h. An enhanced chemiluminiscent ECL system (GE Healthcare, Little Chalfont, UK) was applied according to the manufacturer’s protocol. All experiments were performed in triplicate. Scanning densitometry of blots was analyzed using QuantiScan software (Biosoft, Cambridge, UK).