Bicinchonic acid assay

The L02 cells or middle right liver tissues of rats were lysed in lysis buffer (Genechem Co., Ltd., Shanghai, China). Lysates were centrifuged at 7,500 × g for 10 min at 4°C, and the supernatant was collected. Total protein levels in supernatant samples were quantified using a bicinchonic acid assay (Thermo Fisher Scientific, Inc.). Samples (50 µg protein) underwent 10% SDS-PAGE. Proteins were then electroblotted onto a polyvinylidene difluoride membranes. The membrane was blocked with 5% fat-free milk at 4°C overnight, followed by incubation with rabbit eNOS primary antibody (1:1,000; cat. no. MAB9028; R&D Systems, Inc., Minneapolis, MN, USA) at 37°C for 2 h. Membranes were washed three times with TBS-T, then incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibodies (1:2,000; cat. no. sc-2065; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The density of the corresponding bands was measured quantitatively using Image-Pro Plus software, version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and corrected by reference to the value for GAPDH (1:1,000; cat. no. NB300-221; R&D Systems, Inc.).

All cells were briefly rinsed with cold PBS prior to collection. Both whole cells and subcellular fractions were lysed in RIPA buffer (50 mM Tris at pH 7.5, 150 mM NaCl, 0.5% SDS, 0.5% sarkosyl, 0.5% NP40, 20 mM EDTA, Roche protease inhibitors, 1 mM PMSF) on ice. Protein concentrations of lysates were determined by the bicinchonic acid assay (Thermo Fisher) before being analyzed by SDS-PAGE. The primary antibodies used included anti-CARM1 (Cell Signaling Technology, 3379), anti-skp2 (Santa Cruz Biotechnology, sc-7164), anti-LC3B (Cell Signaling Technology, 3868), anti-LAMP1 (Cell Signaling Technology, 3243), anti-C9orf72 (Bio-Rad VMA00065), anti-PARP (Cell Signaling Technology, 9542), anti-β-actin (Santa Cruz Biotechnology, sc-47778), anti-NOX2 (Abcam, ab129068), anti-ubiquitin (Cell Signaling Technology, 3936), anti-calnexin (Cell Signaling Technology, 2679P), anti-ACC (Cell Signaling Technology, 3676), and anti-ADRP (Progen Biotechnik, GP42).