Total protein was extracted using RIPA lysis buffer ( R0020, Solarbio Technology, Beijing, China) containing Phosphatase Inhibitor and Protease Inhibitor Cocktail ( C0001,MCE HY-K0023, Targetmol, MA, USA) at 4°C. About 10 µg of total protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane ( ISEQ00010, Merck KGaA, Darmstadt, Germany). After blocking in the blocking buffer containing 5% skimmed milk powder, the membranes were incubated with the rabbit anti-human KDF1 antibody ( PA5-55926, Thermo Fisher Scientific, MA, USA, dilution 1:1000) or mice anti-human GAPDH antibody ( YM3029, Immunoway, TX,USA, dilution 1:1000), overnight at 4°C. Then the membrane was washed and incubated with HRP-conjugated goat anti-rabbit ( B0201, Immunoway, TX, USA) or rabbit anti-mouse ( B0101, Immunoway, TX, USA) antibody for 2 h at 37°C. The immunolabeled proteins were detected by chemiluminescence using the Chemiluminescent hRP substrate (Merck KGaA, Darmstadt, Germany). Densitometric analysis was performed using the 1.52a version Image J software (National Institutes of Health, MD, USA).

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