Antibody dilution buffer

Total protein concentration was calculated by the BCA Protein Assay kit (Pierce, Rockford, IL, USA), according to the manufacturer's instructions. The cells and tissues were lysed in cell lysis buffer for 20 min vibration on the ice and centrifuged at 12.000 × g at 4°C for 15 min. Proteins were separated upon 10% SDS-PAGE and transferred onto 0.45 mm PVDF membrane (Bio-Rad, California, Hercules, USA). The membranes were incubated with 5% nonfat milk powder which dissolved in TBST (20 mM Tris-HCL, pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 2 hours. The following protein antibody Nrf2 (68.0 kDa), Histone H3 (15.0 kDa), and GAPDH (40.2 kDa) (Cell Signaling Technology, CST); HO-1 (34.6 kDa), NQO-1 (30.0 kDa), and GST (24.0 kDa) (Abcam); and ANXA5 (36.0 kDa) (Santa Cruz, CA) were used to bind with corresponding proteins and incubated overnight at 4°C. All these protein antibodies were diluted by the Antibody Dilution Buffer (Beyotime, China) with a ratio of 1 : 1000-2000. HRP-conjugated secondary antibody (1 : 4000, Cell Signaling Technology, USA) was used to bind with primary antibodies for about 1.5 hours. Finally, immunoreactive bands were visualized with electrochemiluminescence reagent (Amersham, Uppsala, Sweden). Densitometry and quantitative analysis were analyzed using the ImageJ software (Bethesda, USA) and regulated by Histone H3 and GAPDH.

Cells and tissues were collected and mixed with PMSF (Beyotime, China) containing protease inhibitor. The total protein was extracted using the ExKine™ Pro Animal Cell/Tissue Total Protein Extraction Kit (Abbkine, USA). BCA protein concentration assay kit (Beyotime, China) was used to determine total protein concentration. The extracted protein (20 μg per well) was separated with 8-10% SDS-PAGE gel and transferred to the PVDF membrane (Merck Millipore; Billerica, MA, USA). The membranes were blocked with QuickBlock™ for 1 hour and incubated with primary antibodies overnight at 4°C. Then, membranes were incubated with secondary antibodies (dissolved in antibody dilution buffer (Beyotime, China) for 1 hour at room temperature and then assessed using chemiluminescence (Abbkine, California, USA).
Primary antibodies used in this study were MCT4 (proteintech, 22787-1-AP), Vinculin (proteintech, 26520-1-AP), SLC7A11/xCT (proteintech, 26864-1-AP), AIFM2/FSP1 (proteintech, 20886-1-AP), pAMPKα (cst, 50081), ACC (cst, 3676) and LC3A/B (cst, 12741) and Tubulin (proteintech, 11224-1-AP). The dilution ratios of the above primary antibodies were 1 : 1000. Secondary antibodies used in this study were HRP Goat Anti-Mouse IgG (H+L) (abclonal, AS003) and HRP Goat Anti-Rabbit IgG (H+L) (abclonal, AS014). The dilution ratios of the above secondary antibodies were 1 : 5000.

Cells were lysed with RIPA lysis buffer (Solarbio, Beijing, China) containing 1 × PMSF (Solarbio, Beijing, China) to extract total protein.
Nuclear and cytoplasmic proteins were extracted separately with the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Jiangsu, China). Protein lysates (20 μg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (0.45 μm; Millipore, Burlington, MA, USA). The membranes were incubated with antibodies against β–catenin (1 : 4000 in antibody dilution buffer (Beyotime, Jiangsu, China), the following antibodies used the same dilution buffer), C-myc (1 : 1000), Cyclin D1(1 : 1000), GAPDH (1 : 5000), Wnt5a (1 : 1000), β-tubulin (1 : 2000) (Abcam, Cambridge, UK), and Histone (1 : 2000) (Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight and then incubated with corresponding horseradish peroxidase- (HRP-) conjugated second antibodies (1 : 1000) (Cell Signaling Technology, Danvers, MA, USA) at 37°C for 1 h. The bands were visualized with enhanced chemiluminescence (Millipore, Burlington, MA, USA). The intensity of each band was calculated using Image J software (National Institutes of Health, MD, USA).

The cells were lysed with NETN buffer (20 mM Tris-HCl at pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40 (56,741, Sigma-Aldrich)) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific). The lysate protein concentration was measured using the BCA protein assay kit (Pierce). After equalization, 10 µg of each protein sample was processed via SDS-PAGE, transferred to PVDF membranes, and blocked using 5% non-fat milk (232,100, BD Biosciences) diluted in 1× Tris-buffered saline supplemented with 0.5% Tween-20 (TBST). These membranes were incubated overnight at 4 °C with primary antibodies, followed by their HRP-labeled secondary counterparts (W4011 for rabbit and W4021 for mouse originated primary antibodies, Promega). The immunoreactive bands were visualized by the enhanced chemiluminescence (ECL). The primary antibodies were diluted with Antibody Dilution Buffer (P0023A, Beyotime) and the secondary antibodies were diluted in 1× TBST. GAPDH was used as the control. Band quantification was done via ImageJ.

Cells and tissues were collected and lysed with RIPA lysis buffer (Thermo Fisher Scientific) supplemented with the proteinase inhibitor PMSF (Solarbio, Beijing, China) at a ratio of 100:1 (v/v). Protein concentration was determined with the BCA Protein Assay Kit (Beyotime). Equal quantities (20 ug) of protein extracts were separated with 10% SDS-PAGE and transferred to PVDF membranes (Merck Millipore; Billerica, MA, USA). The membrane was blocked with skimmed milk for 1 h and incubated with primary antibodies overnight at 4 °C.Antibodies are listed in Supplementary “Materials and Methods”. For detection, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (ZSGB-BIO) dissolved in antibody dilution buffer (Beyotime) for 1 h at room temperature. The membranes were visualized with chemiluminescence (Bio-Rad; Hercules, CA, USA) according to the manufacturer’s protocol.