Western blot assay

HT-29, HCT-116, SW480, SW620, SW-1116 or transfected and null SW620 colorectal cancer cells were harvested 48 h following transfection, during the logarithmic growth phase, and total cellular protein was extracted. All cells were washed three times with PBS, centrifuged at 1,000 × g for 5 min at room temperature, and re-suspended in radioimmunoprecipitation assay lysis (Beijing Solarbio Science & Technology, Beijing China) and extraction buffer containing 1 mM phenylmethylsulfonyl fluoride protease inhibitor (Beijing Solarbio Science & Technology) on ice for 30 min to ensure completely lysis. A total volume of 5 ml 5X loading buffer [(250 mM Tris-HCL (pH 6.8), 10% (w/v) SDS, 0.5% (w/v) BPB, 50% (v/v) glycerin and 5% (w/v) β-mercaptoethanol)] was added and boiled at 100°C for 5–10 min. then centrifuged at 12,000 × g for 15 min at 4°C to collect the supernatant. The total protein concentration was detected using the bicinchoninic acid method (BCA) (Beyotime Institute of Biotechnology, Haimen, China). A total of 40 µg protein for each sample was uploaded and separated by SDS-PAGE, with a 5% stacking gel and 12% separating gel, and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were then blocked with TBS containing 0.05% Tween-20 (TBST) and 5% non-fat milk for 1 h at room temperature. Polyclonal rabbit anti-β actin (dilution, 1:200), rabbit monoclonal anti-ARPC4 (dilution, 1:200), polyclonal rabbit anti-vimentin antibody (dilution, 1:200), rabbit polyclonal anti-E-cadherin antibody (dilution, 1:200) and PCNA rabbit polyclonal antibody (dilution, 1:200) antibodies were added to incubate at 4°C overnight. Following washing three times for 10 min each with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (dilution, 1:5,000; cat no. A8275; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and peroxidase-conjugated goat anti-rabbit (dilution, 1:5,000; cat no. A0418; Sigma-Aldrich; Merck KGaA), goat anti-rabbit IgG-HRP secondary antibody (dilution, 1:5,000; cat no. sc-2370; Santa Cruz Biotechnology, Inc.), goat anti-rabbit IgG-HRP secondary antibody (dilution, 1:5,000; cat no. sc-2030; Santa Cruz Biotechnology, Inc.) and chicken anti-rabbit IgG-HRP secondary antibody (dilution, 1:5,000; cat no. sc-2955; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Subsequent to washing with TBST, the membranes were developed using an enhanced chemiluminescence western blot detection system (Merck KGaA). Quantity One 4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and Image J software (version 2.1; National Institutes of Health, Bethesda, MD, USA) was used analyze the results and calculate the expression of ARPC4 and β-actin. β-actin was used as a reference protein.