Gel pro analyzer 4

Cells were placed on ice immediately following treatment and washed with ice-cold Hank’s Balanced Salt Solution. Nuclear proteins were prepared using the CellLytic NuCLEAR extraction kit (Sigma-Aldrich). All the wash buffers and final resuspension buffer included a 1× protease inhibitor cocktail (Pierce, Rockford, IL, USA), NaF (5 mM), and Na3VO4 (200 mM). The protein concentration of the lysate was measured using the BCA protein assay kit (Thermo Fisher). Nuclear or total cell proteins were resolved on 8 to 12% SDS-PAGE and transferred by electroblotting to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% bovine serum albumin reagent (Beyotime, Jiangsu, China) and incubated overnight at 4 °C with primary antibody dilution buffer (the dilution followed the specification; Abcam) and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000) for 2 h. Afterward, the membranes were developed using the enhanced chemiluminescence substrate LumiGLO (Millipore, Bedford, MA, USA) and exposed to X-ray film. The bands were analyzed with Gel-Pro Analyzer 4.0 (Bio-Rad, Hercules, CA, USA).

The expression of the wild-type and variant SLCO1B1 and SLC22A1 proteins was detected with the tag anti-EGFP mAb (Sangon Biotec, Shanghai, China) according to the typical procedure after the plasmids were transfected into the HEK293 cells. A bicinchoninic acid protein assay was used to quantify the total protein in the samples. The quantitative analysis of the integral OD of the bands in the western blot was performed using a Gel-Pro analyzer 4.0 (BIO-RAD, USA).

Western blot analyses were performed on cell lysate prepared from three esophageal cancer cell lines and esophageal tissue as described previously. Equal amount of protein lysate were loaded and separated on 10 % SDS–polyacrylamide gel electrophoresis and then transferred to a PVDF membrane (Millipore, USA). After being blocked by 5 % fat-free milk in TBS buffer, membranes were probed with the following antibodies overnight at 4 °C: mouse monoclonal anti-PFN2 (Abcam; 1:1000 dilution), rabbit anti-E-cadherin (Santa Cruz; 1:200 dilution), rabbit anti-Vimentin (Abcam; 1:1000 dilution), rabbit anti-Snail (Proteintech; 1:500 dilution), rabbit anti-Slug (Proteintech; 1:200 dilution), rabbit anti-ZEB1 (Proteintech; 1:500 dilution) and mouse anti-β-actin (Zhongshan Biotechnology; 1:1000). Bound antibodies were detected with secondary HRP-conjugated antibodies (Santa Cruz) for 2 h at room temperature. After washing, the resulting bands were visualized using the standard ECL procedure (Kangwei, Beijing). Images were acquired using the image acquisition system (BioRad, USA) and their grayscale value was analyzed by the image analysis program (Gel-Pro Analyzer 4.0, USA).