Radioimmunoprecipitation assay protein extraction reagent

CRC tissues and cells were lysed with radioimmunoprecipitation assay protein extraction reagent (Beyotime Institute of Biotechnology, Haimen, China) supplemented with 1% phenylmethanesulfonyl fluoride (Seebio Science & Technology Co., Ltd., Shanghai, China). Protein concentrations were measured using an enhanced BCA protein assay kit (Beyotime Institute of Biotechnology). Each lane was loaded with an equal amount of protein (30 µg), and proteins were then separated using 8% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% milk for 2 h at room temperature. The membranes were then placed in a TBS and Tween-20 (TBST) solution containing anti-ERCC6L antibody (1:1,000; cat. no. BC008808; Wuhan Sanying Biotechnology, Wuhan, China) and were allowed to react at 4°C overnight for ~12 h. The following day, following washing with TBST, the blots were incubated with a horseradish peroxidase-labeled anti-rabbit secondary antibody (1:5,000; cat. no. GB233303-1; Servicebio, Woburn, MA, USA) for 2 h at room temperature. The blots were visualized using a Super ECL detection reagent (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Each set of analyses was repeated a minimum of three times.

Cells were collected and lysed using radioimmunoprecipitation assay protein extraction reagent (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (Roche, Pleasanton, CA, USA). The protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Approximately 50 μg of protein extract was separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, transferred to nitrocellulose membranes (Sigma, St. Louis, MO, USA) and incubated with specific antibodies. Enhanced chemiluminescence chromogenic substrate was used to visualize the bands, and the band intensities were quantified by densitometry (Quantity One software; Bio-Rad) using GAPDH as the control. Antibodies (1:1,000 dilution) against E-cadherin and N-cadherin were purchased from BD (BD Biosciences), and antibodies against vimentin, matrix metalloproteinase (MMP)-2 and MMP-9 were purchased from Cell Signaling Technology.