Anti ogg1

Liver mitochondria were isolated in a medium containing 220 mM mannitol, 70 mM sucrose, 20 mM Tris–HCl, 1 mM EDTA, and 5 mM EGTA, pH 7.4, at 4 °C according to [16 (link)]. In addition, 10 μg of mitochondrial proteins were used for Western immunoblotting analysis. Anti-TFAM (1:50,000), anti-VDAC (1:50,000, Abcam, Cambridge, UK), anti-OGG1 (1:2500, Abcam, Cambridge, UK), anti-APE1 (1:5000, Abcam, Cambridge, UK), anti-MFN2 (1:5000, Abnova, Taipei, Taiwan), anti-DRP1 (1:2500, Abnova, Taipei, Taiwan), anti-Cyt c (1:500, Pharmingen, San Diego, CA, USA), and anti-Lon Protease (1:10,000) were used as primary antibodies. The antibodies against TFAM and Lon were custom-made and kindly donated, respectively, by Dr. H. Hinagaki (Department of Chemistry, National Industrial Research Institute of Nagoya, Nagoya-shi, Aichi, Japan) and Dr. C. Suzuki (Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA). The proteins were detected by chemiluminescence and immunoreactive bands were quantified using the Image Lab Software (BioRad Laboratories Inc., Hercules, CA, USA) and normalized against VDAC-expression.

The lung tissues or AECII cells were homogenized in radioimmunoprecipitation assay lysis buffer with phenylmethanesulfonyl fluoride (Sigma-Aldrich). Following centrifugation at 15,000 × g for 10 min at 4°C, the protein lysates in the supernatant were quantified using a Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology, Shanghai, China). The proteins were loaded and separated through a 10% SDS-polyacrylamide gel and were transferred onto polyvinylidene fluoride membranes (Merck Milipore, Boston, MA, USA). The membranes were blocked in 5% non-fat milk dissolved in Tris-buffered saline (TBS) for 2 h at room temperature prior to incubation overnight at 4°C with anti-OGG1 (1:400; Abcam) and anti-β-actin primary antibodies (1:1,000; Santa Cruz Biotechnology, Inc.). The membranes were then washed in TBS-0.2% Tween 20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibody at 37°C for 2 h. The membranes were washed in TBST, as previously. An enhanced chemiluminescence detection kit (EMD Millipore, Billerica, MA, USA) and a WD-9413B Gel Imaging system (Liuyi Instrument Factory, Beijing, China) were used for chemiluminescence analysis and imaging. The bands were quantified using ImageJ 1.45s software (National Institutes of Health, Bethesda, MD, USA) and the optical densities of all bands were normalized to those of β-actin.

Protein extract (20 μg) was applied on 10% polyacrylamide gel. Later, proteins in the gel were transferred to a nitrocellulose membrane for Western blotting (Towbin et al., 1979). The membrane was blocked for 1 h with 5% bovine serum albumin (BSA; Sigma-Aldrich) at room temperature and subsequently incubated with the primary antibody PBS-T (PBS, 0.1% tween) with 5% BSA (Sigma-Aldrich), for 16 h at 4 ^oC. The primaries antibodies used were anti-PARP1 (1:1000; Abcam; ab6079), anti-γH2Ax (1:1000; Cell Signaling), anti-NRF2 (1:500; Abcam), anti-p-NRF2 (1:1000; Abcam), anti-OGG1 (1:1000; Abcam), Anti-pan-ADP-ribose binding reagent (1:1000; Merck) and anti-histone 3 (Abcam; 1:5000). Then, membranes were placed in PBS-T solution with 5% milk, and secondary anti-mouse or rabbit IgG peroxidase antibody (Sigma-Aldrich) diluted 1:5000. The membrane was developed with chemidoc (UVITEC) in the presence of Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare). Alternatively, membranes were stripped with Restore Western Blot Stripping Buffer (ThermoFisher) and reincubated with another primary and secondary antibody. Band quantification was performed in ImageJ (v 2.1.0). Briefly, the area from relative density of each band from target protein was measured and normalized with endogenous control.