0.45 mm polyvinylidene fluoride membranes

Whole protein lysates were prepared with RIPA lysis buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) according to the manufacturer's instructions. SDS‐PAGE electrophoresis was performed using Novex 4–20% Tris‐Glycine gels (Life Technologies), and Western blots were performed using 0.45 mm polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). Primary antibodies, KDM6B (1:1000), β‐actin (1:10,000), were purchased from Cell Signaling Technology (Daners, MA, USA). The secondary antibodies were horseradish peroxidase‐linked goat‐anti‐rabbit IgG (Santa Cruz Biotechnology). Blots were visualized using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, Bilerica, MA, USA).

Total proteins were extracted from 21 paired fresh frozen GC specimens and corresponding adjacent normal gastric specimens using RIPA lysis buffer and PMSF (Beyotime, China). The protein concentration was detected by BCA protein assay kit (Beyotime, China). Subsequently, an equivalent amount of protein of each paired specimen was separated by SDS-PAGE on 10% polyacrylamide gels and then was electrotransferred to 0.45 mm polyvinylidene fluoride membranes (Millipore, USA) for 1 h at 200 ma. After blocking in 5% nonfat milk diluted with TBST (tris-buffered saline/Tween-20) for 1 h at room temperature, the membranes were separately incubated with rabbit anti-ILF2 monoclonal antibody (1 : 3000; Abcam) and rabbit anti-GAPDH antibody (1 : 3000; Bioss) at 4°C overnight. On the second day, after washing 3 times with TBST per 10 min, the membranes were incubated with peroxidase-conjugated AffiniPure goat anti-rabbit IgG (1 : 6000; zsgb) for 1 h at room temperature. Finally, after washing 3 times with TBST per 10 min, the targeted protein was detected with the enhanced chemiluminescence system according to the manufacture's instruction. The intensity of ILF2 protein band was quantified by ImageJ software and normalized with GAPDH.

Total protein was extracted in cells or kidney tissue using RIPA (Beyotime Biotechnology, Shanghai, China) and quantified using the BCA (Thermo Scientific, Waltham, MA, USA) assay kit. Equal amounts of protein from each sample were electrophoresed on 10% Tris-glycine sodium dodecyl sulfate-polyacrylamide gels, and western blots were transferred to 0.45-mm polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). Membranes were blocked with TBST containing 5% nonfat dry milk for one hour at room temperature and then incubated with primary antibodies against mouse fibronectin (ab45688, ABCAm), vimentin (5741 P, CST), N-cadherin (22018-1-AP, ProteinTech), β-catenin (ab32572, ABCAm), snail (3879 P, CST), twist (sc-81417, Santa Cruz) or GAPDH (#5174, CST) overnight at 4 °C, followed by incubating with a horseradish peroxidase-conjugated secondary antibody. Densitometric analysis results were standardized as GAPDH expression and expressed as fold change relative to those of negative control.