Enhanced chemiluminesence

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).

The expression of Nrf2 nucleoprotein, total protein, and HO-1 in the hippocampal tissue was assayed with the Western blotting technique^{8 (link)}. The hippocampus tissues were obtained 24 h after operation and stored at – 80 °C. Protein extraction was performed by following the instructions of the total protein and nuclear protein extraction kit (Thermo, USA), and protein quantification was conducted with a BCA Protein Assay kit (Solaibo, China). The protein samples were divided in equal amounts using the sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After 30 μg protein was added to the gel, the membranes were blocked with 5% skim milk TSB buffer for 2 h before being incubated with primary antibodies (Nrf2, 1:1000; GAPDH, 1:5000; HO-1, 1:1000; PCNA, 1:5000, Sanying, Wuhan, China) overnight at 4 °C. After five washes with TBST, the membranes were incubated with goat anti-mouse secondary antibody (1:10,000, Sigma, USA) at room temperature for 2 h. The enhanced chemiluminesence (Bio-Rad, USA) was used to observe the blots following the manufacturer’s instruction, and the quantification of the band relative density was measured using the densitometry (Molecular Analyst Image-analysis Software, Bio-Rad, USA).

The expression of HO-1, Nrf2 total protein and Nrf2 nucleoprotein of the lung samples were analyzed by Western blot technique. The tissues stored at −80°C were homogenized in 13.2 mmol/L Tris-HCl, 5.5%glycerol, 0.44%SDS and 10% β-mercaptoethanol. The proteins were extracted according to the instructions of the total protein and nuclear protein extraction kit (Thermo, USA), while the protein concentration was detected on the basis of the BCA protein assay kit (Thermo, USA). Equal amounts of soluble protein were fractionated by 12% SDS-PAGE and were transferred to a PVDF membrane (Bio-Rad, USA). Blots were washed triple for 5 min in TBS and then were incubated overnight at 4°C with polyclonal rabbit antibodies against HO-1 (1∶800, Abcam, UK) or Nrf2 (1∶300, Biorbyt, UK). Primary antibodies were diluted in 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, blots was incubated at 37°C for 1 h with the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1∶3000 dilation, Biorbyt, UK). The blots were visualized with the enhanced chemiluminesence (Bio-Rad, USA) according to the manufacturer’s instruction [26] (link), and the relative density of bands was quantified by densitometry (Molecular Analyst Image-analysis Software, Bio-Rad, USA).