2 2 2 trichloroethanol

Trichloroethylene (99.9 % purity) and hydrogen peroxide were obtained from Fisher Scientific (Waltham, MA, USA) while trichloroacetic acid (≥ 99.0% purity), 2,2,2-trichloroethanol (≥99% purity), forskolin (≥ 98.0% purity), mifepristone (RU486) (≥98% purity), hydroxyflutamide (OHF) (≥98% purity), and oxalic acid (≥99% purity) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All test chemicals (Table S1) were diluted into dimethyl sulfoxide (DMSO) (≥99.7% purity) from Fisher Scientific (Waltham, MA, USA). Dihydrotestosterone (DHT) mimic, Cl-4AS-1, (>99% purity), 17β-estradiol (E2) (>99% purity), and tamoxifen (TAM) (≥99% purity) were acquired from Tocris Bioscience (Minneapolis, MN, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was obtained from Amresco (Fountain Parkway Solon, OH, USA).

Protein samples (10–60 µg) from ethanol-fed and pair-fed controls were separated using 12% SDS-PAGE at 150 V for 1 hour, then transferred to activated Hybond-PVDF membrane (GE Healthcare, Buckinghamshire, UK). Membranes were blocked using 5% (w/v) non-fat dry milk in Tris-Buffered Saline-Tween 20 TBS/0.1 % (v/v) Tween (TBS-T) for 1 hour at room temperature. Membranes were incubated with primary antibodies against SOD2 (ab13534), SOD2 acetyl K68 (ab137037), or SOD2 acetyl K122 (ab214675) (Abcam, Cambridge, MA) overnight at 4°C, then washed three times with TBS-T, and incubated with horseradish peroxidase conjugated secondary antibody at room temperature for one hour. Membranes were washed again with TBS-T three times and then Clarity Western ECL Substrate (BioRad) was applied before imaging via Chemidoc® MP (Bio-Rad, Hercules, CA). 2,2,2-trichloroethanol (Sigma, Saint Louis, MO) stain was used to visualize overall protein load.
Two-dimensional SDS-PAGE was performed using 200 µg of liver whole cell extract and was separated on IPG strips (pH 3–11) (Bio-Rad), then resolved using 10% SDS-PAGE. The proteins were then transferred onto PVDF membrane and probed with anti-SOD2 (ab13534) and SOD2 acetyl-K68 (ab137037) antibody (Abcam, Cambridge, MA) using standard Western blot analysis as described above.