Ecl substrate buffer

The expression of proteins associated with cell cycle progression, apoptosis, TP53 targets p53 and p21, MAPK signaling and epithelial-mesenchymal transition (EMT) was evaluated using western blot analysis. Total protein content in HepG2 cells was extracted using a radioimmunoprecipitation assay lysis buffer (Bioswamp; Wuhan Beinglay Biotech Co., Ltd.) containing protease and phosphatase inhibitors. Proteins were quantified using the BCA kit (Bioswamp; Wuhan Beinglay Biotech Co., Ltd.). A total of 10 µg proteins were separated using SDS-PAGE (12%) and transferred onto PVDF (EMD Millipore). The membranes were then blocked with 5% skimmed milk for 2 h at room temperature and incubated with primary antibodies overnight at 4˚C. After washing, the membranes were incubated with secondary antibody for 1 h at room temperature. Immunoreactivity was visualized by colorimetric reaction using ECL substrate buffer (EMD Millipore). The membranes were then detected by an automatic chemiluminescence analyzer (Tanon-5200; Tanon Science and Technology Co., Ltd.) and the band gray values were read using TANON GIS 4.2 software (Tanon Science and Technology Co., Ltd.). All experiments were performed in triplicate. Detailed information of all antibodies used in the present study are presented in Table I.

Hep3b cells were washed in PBS and lysed using the protein extraction reagent RIPA (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration of proteins was measured by BCA kit (cat. no. ab102536; Abcam). Equivalent amounts of proteins (30 µg) from each sample were electrophoresed on SDS-polyacrylamide gel (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane, blocked in 4% skim milk for 2 h at room temperature and incubated with the following specific primary antibodies: E-cadherin antibody (ab219332 1:1,000; Abcam), α-catenin antibody (ab51032 1:2,000; Abcam), N-cadherin (ab76011, 1:5000 dilution, Abcam), vimentin (ab92547 1:1,000; Abcam), p-PI3K (ab182651 1:1,000; Abcam), PI3K (ab227204 1:1,000; Abcam), p-AKT (ab38449, 1:500 dilution, Abcam), AKT (ab18785 1:1,000; Abcam), VEGF (ab214424 1:1,000; Abcam), VEGFR2 (ab221679 1:1,000; Abcam), Snail (ab53519 1:1,000; Abcam), Slug (ab27568 1:1,000; Abcam) and MPP9 (ab38898 1:1,000; Abcam) overnight at 4°C. β-actin (ab8277 1:1,000; Abcam) was used as internal reference. Then, the membranes were incubated in HRP-linked goat anti-rabbit IgG secondary antibody (ab97051; 1:10,000; Abcam) for 2 h at room temperature. Immunoreactivity was visualized by a colorimetric reaction using an ECL substrate buffer (EMD Millipore) and membranes were scanned with Gel Doz EZ imager (Bio-Rad Laboratories, Inc.).