Anti gapdh

For total protein analysis, cells were lysed in ice-cold RIPA buffer supplemented with 1.0 mM PMSF. For liver tissue homogenates from mice, the liver was dissected into small sections (40 mg) and homogenized in ice-cold RIPA buffer, also containing 1.0 mM PMSF. After incubating on ice for 40 min, we obtained the proteins by centrifugation at 12,000 g for 20 min at 4°C. Protein concentrations were determined using the BCA protein assay kit. Equal amounts of protein (approximately 30.0 μg) were loaded onto SDS-PAGE gels, and the proteins were transferred to a PVDF membrane (IPVH00010, Millipore) via electroblotting.
We used the following primary antibodies: anti-phospho-IKKβ (#2697, 1:1000), anti-NF-κB-p65 (#8242, 1:1000), anti-phospho-NF-κB-p65 (#3033, 1:1000), anti-Akt (#4691, 1:1000), anti-phospho-Akt (Ser473, #2859, 1:1000), anti-phospho-GSK3β (#5558, 1:1000), anti-AMPKα (#5831, 1:1000), anti-phospho-AMPKα (#2535, 1:1000), anti-Col1a1 (#72026, 1:1000), and anti-α-SMA (#19245, 1:1000) from Cell Signaling Technology; anti-IKKβ (#ab124957, 1:1000) from Abcam; anti-GSK3β (#22104-1-AP, 1:1000), anti-FASN (#10624-2-AP, 1:1000), anti-ACLY (#67166-1-Ig, 1:1000), anti-PPARγ (#16643-1-AP, 1:500), anti-LKB1 (#10746-1-AP, 1:500), anti-GAPDH (#60004-1-Ig, 1:2000), and anti-HRP-coupled secondary antibody (#SA00001-1 or #SA00001-2, 1:5000) from Proteintech; anti-Ac-Lys (#sc-32268, 1:100) from Santa Cruz Biotechnology; and anti-SREBF1 (#557036, 1:1000) from BD Biosciences.
After overnight incubation with the primary antibodies at 4°C, the membranes were incubated with the HRP-coupled secondary antibody. We detected the protein bands using the Tanon chemiluminescent substrate system (Tanon).

The protein was extracted from total cells or tissues using RIPA buffer containing PMSF and phosphatase inhibitor as previously described (Li et al., 2019a (link),b (link),c (link),d (link),e (link); Liu et al., 2019a (link), b (link); Lu et al., 2019 (link); Shao et al., 2019 (link); Weng et al., 2019 (link); Yang et al., 2019a (link), b (link); Zhao et al., 2019 (link)). 30 μg of protein were loaded in each lane and separated by 8% PAGE-SDS gel with all-blue protein markers (Bio-Rad). Proteins were transferred to nitrocellulose membranes (Bio-Rad) in a Mini-Trans-Blot Cell (Bio-Rad). The membranes were blocked with 5% fat-free milk powder in Tris-buffered saline at room temperature for half an hour and then incubated with the following primary antibodies at 4°C overnight: Western blot analyses were performed with anti-p16 (1:1,000, Proteintech, catalog# 10883-1), anti-Collagen type I (1:1,000, Proteintech, catalog# 14695-1), anti-α-SMA (1:5,000, Abcam, catalog# ab5694), anti-GAPDH (1:2,000, Proteintech, catalog# 60004-1), anti-p38 (1:1,000, Proteintech, catalog# 14064-1), and anti-phospho-p38 antibodies (1:2,000, Cell Signaling, catalog# 1682). Image J software was used for densitometrical quantification and densities of target proteins were normalized to those of β-actin. Data are expressed as relative protein levels compared to the control group which is arbitrarily set as 1.

Total proteins were extracted by lysing cells in 6‐well plate with 60 μL RIPA buffer supplemented with protease inhibitors (#G2006, Wuhan Goodbio Technology) and phosphatase inhibitors (#G2007, Wuhan Goodbio Technology) and quantified by BCA protein assay kit (#P0011, Beyotime). One microgram of proteins was separated by 10% SDS‐PAGE and transferred to PVDF membrane (#IPVH00010, Merck Millipore). The membrane was blocked with 5% Non‐Fat Milk (#A600669, Sango Biotech) for 1 hour at room temperature and incubated with specific antibody at 4°C overnight followed by HRP‐conjugated secondary antibody incubation for 1 hour at room temperature. Images of membrane with proteins were taken with imager (Bio‐rad ChemiDoc MP, Bio‐rad). The antibodies are recorded: anti‐GAPDH (#60004‐1‐lg, Proteintech), anti‐YTHDF2 (#24744‐1‐AP, Proteintech), anti‐E‐cadherin (#20874‐1‐AP, Proteintech), anti‐N‐cadherin (#66219‐1‐AP, Proteintech), anti‐VIMENTIN (#10366‐1‐AP, Proteintech), anti‐MMP9 (#10375‐2‐AP, Proteintech), anti‐MMP2 (#10373‐2‐AP, Proteintech), anti‐SNAIL (#13099‐1‐AP, Proteintech), anti‐CDK6 (#14052‐1‐AP, Proteintech), anti‐CCND1 (#26939‐1‐AP, Proteintech), anti‐CDK4 (#11026‐1‐AP, Proteintech), anti‐KLF4 (#12173S, Cell Signaling Technology), anti‐SLUG (#C1967, Cell Signaling Technology), anti‐METTL3 (#ab195352, Abcam) and anti‐SETD7 (#ab14820, Abcam).

Cells were lysed using a radio-immunoprecipitation assay (RIPA) buffer containing a Protease Inhibitor Cocktail and PhosSTOP Phosphatase Inhibitor Cocktail (both from Roche, Basel, Switzerland) as previously described [43 ]. For immunoprecipitation, cell lysates were incubated with appropriate antibodies and protein G-Sepharose and then subjected to western blotting. The membranes were blotted with appropriate primary antibodies followed by horseradish peroxidase-coupled secondary antibodies and then visualized using chemiluminescence (DNR Bio-Imaging Systems, Jerusalem, Israel). For western blot analysis, β-tubulin was used as a loading control. Antibodies were obtained from various sources, including Sigma–Aldrich (St. Louis, MO, USA; anti-β-tubulin, anti-β-catenin, anti-phosphoserine/threonine, anti-Flag M2 and anti-CCNG2), Cell Signaling Technology (Beverley, MA, USA; anti-p–β-catenin ser33, ser37 and thr42), Abcam (Cambridge, UK; Anti-Phosphoserine, anti-CCNG2 and anti-DACT1), Affinity Biosciences (Changzhou, China; anti-CCNG2), Wanleibio (Shenyang, China; anti-GAPDH), Proteintech Group (Chicago, IL, USA, anti-CCNG2) and Santa Cruz Biotechnology (Santa Cruz, CA, USA; anti-Dvl2, anti-GSK-3β, anti-cyclin G2, anti-DACT1, anti-p-GSK-3β-Ser9 and anti-c-Myc).