Ab45174

IHC was conducted according to the protocol of the Two-step IHC Kit (ZSGB-BIO). Briefly, 4 µm thick slides of paraffin-embedded tissue specimens were processed for antigen retrieval with citric acid buffer (0.01 M, pH 6.0), and endogenous peroxidase activity was quenched with 3% hydrogen peroxide solution. Then, the slides were blocked with 10% bovine serum albumin and incubated overnight at 4 °C with the following primary antibodies: anti-MED15 (1:50; 11566-1-AP; Proteintech), anti-SREBP1 (1:100; AF6283; Affinity), anti-SREBP2 (1:100; ab30682; Abcam), anti-FASN (1:50; DF6106; Affinity), anti-ACC1 (1:100; ab45174; Abcam), anti-ACLY (1:100; ab40793; Abcam), anti-SCD1 (1:100; #2794; CST), anti-PLK1 (1:50; DF7004; Affinity), and anti-Ki67 (1:200, BS-2130R; Bioss). The slides were incubated with biotinylated goat anti-rabbit IgG (1:200) for 1 h at room temperature. DAB (ZLI-0918, ZSBio, China) was used to visualize the immune complexes, and the sections were counterstained with hematoxylin. Immunostaining intensity was measured using ImageJ software (National Institutes of Health, Bethesda, MD) as previously described [56 (link)].

Hepatocytes were harvested and washed twice in ice-cold PBS. Total cellular protein was extracted using a protein extraction kit according to the manufacturer's instructions. Aliquots of 60 μg total protein were denatured for 5 min at 100°C and loaded on 10% sodium dodecyl sulfate polyacrylamide gels and electro-transferred onto polyvinylidene difluoride (PVDF) membranes. After incubation with blocking buffer, membranes were exposed to polyclonal GRP78 (78 kD, 1:250, sc-376768; Santa Cruz, CA), SREBP-1c (68 kD, 1:1000; NB100-2215; Novus, USA), FAS (273 kD, 1:1000; C2065; Cell Signaling, Danvers, MA, USA), SORT1 (85 kD,1:1000; ab16640, Abcam, Cambridge, MA), P65 (65 kD, 1:1000; D14E12; Cell Signaling; Danvers, MA, USA), ACC1 (265 kD, 1:1000; ab45174, Abcam, Cambridge, MA), CHOP (27 kD, 1:1000; L63F7; Cell Signaling, Danvers, MA, USA), PCSK9 (65–80 kD, 1:1000; 85813, Cell Signaling; Danvers, MA, USA), SCD1 (37 kD, Cell Signaling; Danvers, MA, USA). After washing and incubation with secondary antibody, immunoreactive bands were detected by enhanced chemiluminescence (Beyotime, China) solution. The imprinting was exposed with X-ray film to radiograph the spectral band, and the gray value of the spectral band was measured with bandscan software version 5.0 (prozyme Inc, San Leandro, CA). Protein abundance is reported relative to that of β-actin as a ratio of optical densities.

Total protein was extracted using RIPA protein lysis buffer (Beyotime Biotech, Jiangsu, China) with PMSF and complete protease and phosphatase inhibitor cocktail. Protein concentrations were determined by a BSA kit (Beyotime Biotech, Jiangsu, China). Fifty micrograms of protein was separated by gel electrophoresis and transferred to methylcellulose membranes. The membranes were blocked in 5% nonfat dried skimmed milk for 2 hours at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti-HIF-2α (1:1000; #59973; CST), anti-MED15 (1:1000; 11566-1-AP; Proteintech), anti-SREBP1 (1:200; sc-13551; Santa Cruz), anti-SREBP2 (1:500; ab30682; Abcam), anti-FASN (1:1000; ab128870; Abcam), anti-ACC1 (1:1000; ab45174; Abcam), anti-ACLY (1:1000; ab40793; Abcam), anti-SCD1 (1:1000; #2794; CST), anti-PLK1 (1:1000; #4513; CST), Phospho-AKT (Ser473) (1:1000; AF0016; Affinity), AKT (1:1000; AF6261; Affinity), and anti-GAPDH (1:1000, #2118; CST). The membranes were incubated with secondary antibodies for 2 hours after being washed with tris-buffered saline and Tween 20 and were visualized by an enhanced chemiluminescence kit (Biological Industries, Israel).