Cellytic m lysis reagent

Frozen liver tissue (30 mg) was homogenized in 0.5 ml CelLytic M lysis reagent (Sigma-Aldrich) containing 1% phosphatase inhibitor cocktail and protease inhibitor cocktail, and centrifuged at 13,000 × g for 30 min at 4 °C. The protein concentration of the supernatant was determined by the Bradford assay. The cell lysates were separated using SDS-PAGE and transferred onto PVDF membranes, followed incubation with primary antibodies against NF-κB p65, MnSOD, Bax, Bcl-2 (all from Santa Cruz Biotechnology, Dallas, TX), p66^shc, p-p47 phox, p47 phox, cytochrome C (all from Millipore), PAI-1, MCP-1, Cdk2 (all from Abcam), iNOs (BD Bioscience), SIRT1 (AVIVA Systems Biology, San Diego, CA), and GST (Cell Signaling, Danvers, MA). Finally, horseradish peroxidase-conjugated secondary antibodies were used for ECL detection. The results were normalized against β-actin and H1 as internal controls.

Total protein was extracted from the cells in CelLytic M™ lysis Reagent (Merck-Sigma-Aldrich, Darmstadt, Germany). Total protein (60 μg) was transferred to nitrocellulose membrane. The membrane was proved with anti-KDM5B antibody as described in IHC, anti-E2F1 antibody (KH95, Santa Cruz Biotechnology, Dallas, TX), anti-E2F2 antibody (L-20, Santa Cruz Biotechnology) and anti-RB antibody (IF8, Santa Cruz Biotechnology). Anti-Actin (I-19, Santa Cruz Biotechnology) was used as a loading control.

To prepare whole cell lysates, cells were lysed with the CelLytic™M lysis reagent (C2978, Sigma-Aldrich). After thorough mixing and incubation at 4°C for 30 min, lysates were centrifuged at 15,000 g at 4°C for 10 min, and supernatants were collected. To prepare chromatin-bound subcellular fractions, we followed the protocol of Subcellular Protein Fractionation Kit from Thermo Scientific (78840) [8 (link)]. Immunoblotting was carried out using standard procedures.

Cell lysate samples were prepared from the cells lysed with CelLytic M lysis reagent (Sigma-Aldrich) supplemented with complete protease inhibitor cocktail (Roche Applied Science). Whole cell lysates or immunoprecipitated samples were separated by SDS-PAGE and blotted to nitrocellurose membrane. Protein bands were detected by incubating with horseradish peroxidase (HRP)-conjugated antibodies (GE Healthcare) at room temperature for 1 h and visualizing with enhanced chemiluminescence (GE Healthcare).

The liver tissue was lysed in 0.5 mL of CelLytic M lysis reagent (Sigma-Aldrich, St. Louis, MO, USA) with 1% phosphatase inhibitor cocktail and protease inhibitor cocktail and centrifuged at 13,000× g for 30 min at 4 °C. The concentration of protein lysate was determined using the Bradford assay. In addition, the nuclear fraction was harvested using a nuclear extraction kit (Abcam, Cambridge, MA, USA) in accordance with the manufacturer’s instructions. The cell lysates were separated using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes, following incubation with primary antibodies against NF-κB, α-SMA, TGFβ-R1, and TGFβ-R2 (all from Santa Cruz Biotechnology, Dallas, TX, USA). Finally, horseradish peroxidase-conjugated secondary antibodies were added, and the reaction was detected by electrochemiluminescence. The data were calibrated using H1 and β-actin as internal controls.

Whole-cell lysates were prepared from cells lysed with CelLytic M Lysis Reagent (Sigma-Aldrich) supplemented with complete protease inhibitor cocktail (Roche Applied Science) and phosphatase inhibitor. Whole-cell lysates or immunoprecipitated samples were separated by SDS-PAGE and blotted to nitrocellulose membrane. Protein bands were detected by incubating with HRP-conjugated antibodies (GE Healthcare) and visualized with enhanced chemiluminescence (GE Healthcare).

Oxygen consumption of intact cells was determined using a Seahorse Bioscience XF24 Extracellular Flux Analyzer following the manufacturer's instructions. Briefly, cells at a density of ~ 30,000 per well were seeded in XF v7 24‐well plates for 24 hrs. Prior to the measurements, cell medium was replaced with Seahorse XF base medium supplemented with 10 mM galactose, 2 mM Glutamax and 1 mM sodium pyruvate and allowed to equilibrate for 1 hr at 37°C. Oxygen consumption was measured under basal conditions, and in the presence of oligomycin (1 μg/ml), FCCP (0.8 μM), and a combination of rotenone (1 μM) and antimycin A (5 μM) was used to assess the basal and maximal mitochondrial respiration rates. Four baseline measurements were recorded before the consecutive addition of the above‐mentioned compounds, and three response measurements were taken after the addition of each compound. To normalize respiration rates to protein content, cells were lysed with the CelLytic M Lysis reagent (Sigma‐Aldrich) and protein concentrations were measured using the Bradford protein assay (Bio‐Rad, Mississauga, ON, Canada). Basal respiration rate was calculated as oxygen consumption rate (OCR) before oligomycin injection minus OCR after rotenone plus antimycin A injection. Maximal was determined as the OCR after FCCP minus non‐mitochondrial OCR after injection of the ETC. inhibitors.