Anti fto

Quantitative analysis on the change of protein expressions was conducted by western blot analysis according to the previous reports.35 (link), 36 (link) After transfection and induction, the cells were harvested for western blotting analysis. The cells were incubated by radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) at 4°C for 30 min to extract total proteins. Then, approximately 60 μg of total cell lysates was separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membranes were blocked with 5% non-fat milk for 2 h and immunoblotted overnight at 4°C with anti-FTO (Abcam, UK) or anti-β-actin (ZSGB-BIO, China). Membranes were then incubated with secondary antibodies for 1 h at room temperature in a dark room. The blotted bands were visualized using Image Studio software (Alias, USA).

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).