Mab3925

Cells were washed twice with PBS, solubilized in denaturing sample buffer and then subjected to SDS-PAGE. Proteins were electrotransferred to 0.2 µm Protran BA 83 nitrocellulose sheets (Invitrogen, Carlsbad, CA, USA) for immunodetection with the following primary antibodies: H2AX (1:2000; ab20669, Abcam, Cambridge, MA, USA); NRF2 (1:2000; MAB3925, R&D system, Minneapolis, MN, USA); NQO1 (1:3000; ab34173, Abcam, Cambridge, MA, USA); GCLC (1:2000; ab53179, Abcam, Cambridge, MA, USA). Immune complexes were detected with horseradish peroxidase coupled anti-rabbit or anti-mouse IgG antibodies (Amersham^TM, GE Healthcare, Pittsburgh, PA, USA).

The relative amount of various proteins was determined by western blot analysis according to the protocol as described.²⁹ The antibodies used for immunoblot analysis were purchased from Cell Signaling Technologies (CST), Santa Cruz Biotechnology (SCBT) or R&D Biosystems (R&D): anti‐Ubiquitin (SCBT sc8017), anti‐Beclin‐1 (CST 3495), anti‐light chain (LC)‐3B (CST 2775), anti‐GAPDH (CST 2118), anti‐phospho‐p65 (CST 3033), anti‐p65 (CST 8242), anti‐phospho‐p38 (CST 9211), anti‐p38 (CST 9212), anti‐phospho‐AMPK (CST 2535), anti‐AMPK (CST 2532), anti‐TAK1 (CST 4505), anti‐Nrf2 (R&D Systems, MAB3925), and anti‐KEAP1 (CST 8647). HRP conjugated secondary antibodies were procured from Cell Signaling Technology. Band intensities were quantified with Image J software.

We performed protein extraction and western blot analyses as described previously^{20 (link)}. The utilized antibodies included mouse antibody to NQO1 (sc-376023, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Nrf2 (MAB3925, 1:500; R&D System), NFκB (p65) (sc-8008, 1:200; Santa Cruz Biotechnology), goat antibody to HO-1 (AF3169, 1:500; R&D System), β-actin (MAB8929, 1:500; R&D System), goat anti-mouse IgG (sc-2039, 1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and donkey anti-goat IgG (sc-2042, 1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The proteins were visualized using an enhanced chemiluminescence detection kit (Bio-Rad, USA) following the manufacturer’s protocol. Images were analyzed using the Kodak Image Station 2000R (Eastman Kodak Company, Rochester, NY, USA). Protein bands intensity were normalized to β-actin, and the data expressed in terms of percent relative to controls.

Total protein was isolated from liver by homogenization in lysis buffer (0.5 M EDTA, 1 M Tris-HCl pH 8.1, 10% SDS, 10% Empigen,) with 1 × Complete Protease Inhibitor (Roche) and 1 × Phosphatase Inhibitor (Thermo Scientific, Waltham, MA, USA). Samples were further processed with sonication and protein concentrations were determined (BCA assay, Thermo Scientific). Supernatants were collected and filtered using an Amicon Ultra-4 centrifugal filter device (Millipore Sigma) after Western blot analysis. Equal amounts of protein were separated by electrophoresis on 4–20% Mini-PROTEAN TGX gels (BIORAD) and transferred to nitrocellulose membranes. Membranes were blocked (Blocking Buffer, Odyssey^®, Li-Cor, Lincoln, NE, USA) and target proteins were probed with the following primary antibodies: β-actin (Sigma SAB1305546) 1:10,000 dilution, NAD(P)H dehydrogenase [quinone] 1 (NQO1) (Abcam Cat# ab2346, RRID:AB_302995) 1:500 dilution, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) (R&D SYSTEM MAB3925) 1:500 dilution. Membranes were then exposed to an anti-goat, anti-mouse secondary antibody (Odyssey) at 1:10,000 dilution. Proteins were visualized using a Licor Odyssey CLx infrared imaging device and software. All results were repeated at least three times and the results were normalized to a β-actin loading control.