Radioimmunoprecipitation assay buffer

In brief, liver cancer cells were lysed using radioimmunoprecipitation assay buffer (BIOSS) for 30 min on ice. The cell lysate was centrifuged at 12,000 × g for 20 min at 4°C and the supernatant was collected. Protein concentration was determined by BCA protein assay. A total of 30 µg protein/lane was separated by SDS-PAGE on a 10% gel and transferred onto nitrocellulose filter membranes. Following blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C, and then with secondary antibodies for 2 h at room temperature. The primary antibodies used were as follows: Rabbit polyclonal anti-CXCR1 (1:2,000; cat. no. ab137351; Abcam) and anti-CXCR2 (1:2,000; cat. no. ab14935; Abcam) antibodies; monoclonal mouse anti-GAPDH (1:2,000; cat. no. TA-08; Zhongshan Jinqiao Biotechnology) and β-actin (1:2,000, cat. no. TA-09; Zhongshan Jinqiao Biotechnology, Beijing, China). The goat anti-mouse (cat. no. ZB2305; 1:10,000) and anti-rabbit secondary antibodies (cat. no. ZB2301; 1:10,000) were purchased from Zhongshan Jinqiao Biotechnology. The protein bands were visualized by ECL reagents (cat. no. C05-07003; BIOSS). Images were captured and the intensity of the bands was quantitated with the Bio-Rad Versa Doc imaging system (Bio-Rad Laboratories, Inc.).

Total proteins were extracted from the cells and tissues using radio immunoprecipitation assay buffer (BIOSS) mixed with phenylmethylsulfonyl fluoride (BIOSS) and quantified using bicinchoninic acid assay (Beyotime Institute of Biotechnology). Protein extractions (30ug per well) were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (MilliporeSigma). The membranes were blocked with 5% fat-free dried milk for 1 h at room temperature. After incubation with high-affinity anti-FTO (1:1000, Abcam, USA), anti-STAT3 (1:1000, Cell Signaling Technology, USA), anti- phosphorylation-STAT3 (1:1000, Cell Signaling Technology, USA), anti-Bcl-2 (1:1000, Cell Signaling Technology, USA), anti-c-Myc (1:1000, Abways Technology, China), anti-CyclinD-1 (1:1000, Abways Technology, China), anti-β-actin (1:5 000, Bioss, China), or anti-GAPDH (1:5000, Bioss, China) antibodies at 4°C overnight, the membranes were incubated with the HRP-conjugated secondary antibodies goat anti-rabbit (1:10,000; Boster, China) or goat anti-mouse (1:5000; Boster, China) for 1 h at room temperature. Proteins were detected using BeyoECL chemiluminescence kit (Biyuntian, China) and detected using the Amersham ImageQuant 800 system (Cytiva). The density of bands were measured using ImageJ (v1.51, National Institutes of Health).