Catalase

For immunoblot analysis, cell lysates were prepared in 1x RIPA buffer (Thermo Scientific, Waltham, MA) with protease inhibitors (Roche Applied Science). Approximately 25 μg of total protein was analyzed by polyacrylamide gel electrophoresis (PAGE). Primary antibodies were used to detect SOD2 (Abcam; Cat #ab13533, Cambridge, MA), catalase (Cell Signaling, Cat #8841, Danvers, MA), GAPDH (Cell Signaling, Cat #2118), actin (Sigma-Aldrich, Cat #A5441, St. Louis, MO), HO-1 (Enzo Life Sciences, Cat #ADI-SPA-895, Farmingdale, NY), and OXPHOS complexes (Abcam, mouse #ab110413, human #ab13533). Immunoreactive proteins were detected using polyclonal goat anti-rabbit or anti-mouse horseradish peroxidase IgG secondary antibodies (Thermo Scientific, Waltham, MA) and visualized using Supersignal™ chemiluminescent horseradish peroxidase substrate (Thermo Scientific, MA). Densitometry analysis was performed using ImageJ analysis software (National Institutes of Health).

Whole cell lysates for immunoblotting were prepared by dissolving cell pellets in Laemmli Sample Buffer containing 5% 2-mercaptoethanol (Bio-Rad). Antibodies used in this study are as follows: lamin A/C (MAB3211; MilliporeSigma, Burlington, MA), catalase (Cell Signaling Technology, Danvers, MA), Nrf2 (sc-722, Santa Cruz), p16 (sc-468; Santa Cruz), and β-actin (A3854; Sigma-Aldrich, USA).

The Protein Plus Precision All Blue Standards were acquired from Bio-Rad Laboratories (Hemel Hempstead Hertfordshire, UK). The antibodies employed in this investigation were obtained from different manufacturers. The corresponding catalogue numbers from Abcam (Cambridge, USA) were as follows: pSer⁴⁰ Nrf2 (no. ab76026), Nrf2 (no. ab62352), Keap1 (no. ab119403), SOD1 (no. ab16831), Sequestosome 1 (SQSTM1/p62) (no. ab56416) and SQSTM1/p62 (pSer³⁴⁹) (no. ab211324). The antibodies purchased from Cell Signaling Technology (Danvers, MA, USA) were: Thr²⁸⁷ CaMKII (no. 12716), AMPKα (no. 2532), Thr¹⁷² AMPKα (no. 2535), Catalase (no.14097) and SOD2 (no. 13141). Some secondary HRP-conjugated goat anti-rabbit (no. 111-035-144) and the HRP-conjugated goat anti-mouse (no.115-035-003) antibodies were acquired from Jackson ImmunoResearch (West Grove, PA, USA). Other secondary HRP-conjugated goat antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA): anti-rabbit (no. sc2004) and anti-mouse (no. sc2031). See Supplementary Table 1 for a more detailed description of the antibodies and procedures.

Liver homogenates containing 10 μg of whole cell lysate were separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk, the blot was probed with primary antibodies for IGFBP-3, survivin, SOCS-3, NOX4, β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, STAT-3, p-STAT-3, catalase (Cell Signaling Technology, Beverly, MA, USA), and NOX2 (Abcam, Cambridge Science Park, Cambridge, UK).

Proteins from heart tissues or cells were extracted as previously described 18 (link). 30 μg of protein was separated by SDS-PAGE gel, transferred to PVDF membranes. After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies against cytochrome c (Cell Signaling, 1:1000), CHOP (Abcam, 1:1000), catalase (Cell Signaling, 1:1000), α-SMA (Abcam, 1:1000), IκBα (Abcam, 1:1000), NF-κB (Cell Signaling, 1:1000), p-NF-κB (Abcam, 1:500), p65 NF-κB (Abcam, 1:1000), lamin A/C (Abcam, 1:500), p-IKKα (Abcam, 1:500), IKKα (Abcam, 1:1000) at 4°C overnight, and followed by incubation with HRP-conjugated secondary antibodies. Immunoreactive bands were visualized with ECL chemiluminescence and quantified by using Image-Pro Plus software.