Ab238099

Total protein was extracted from HCC cells using RIPA lysis buffer. Extracted proteins were subsequently loaded on SDS–polyacrylamide gels and then transferred to PVDF membranes which were blocked in 5% non-fat milk for 1 h and then incubated with primary antibodies against E-cadherin (1:1000, ab238099, Abcam, USA), N-cadherin (1:5000, Abcam) and GAPDH (1:2000, Abcam) at 4 °C overnight. Then, the membranes were incubated with anti-rabbit secondary antibodies (1:5000; Abcam). Finally, the signals were detected on the ECL chemiluminescence system.

We extracted the total protein with the help of RIPA lysate, then determined with the help of BCA kit, the matched samples were adjusted to the same concentration. After denaturation, we separated the proteins with the help of SDS-polyacrylamide gel electrophoresis and then transferred them onto PVDF membrane. We sealed the PVDF membrane for 1 h using 5% skimmed milk powder, then incubated the PVDF membrane overnight with the following primary antibodies against Bcl-2 (Abcam, ab32124, 1:1000), Bax (Abcam, ab32503, 1:1000), Vimentin (Abcam, ab137321, 1:2000), E-cadhetrin (Abcam, ab238099, 1:100), CyclinD1 (Abcam, ab134175, 1:5000), DNM3 (Abcam, ab134925, 1:1000) protein or GAPDH (Abcam, ab181602, 1:5000). We then incubated the PVDF membrane for 1 h with secondary antibody. ECL kit was used for chemiluminescence detection. The gray value of protein band was calculated using Image-Pro Plus.

The treated HK2s and hRPTECs were washed thrice with PBS and lysed directly using Mem-PER plus membrane protein extraction kit for membrane protein extraction (Cat# 89842, Thermo Fisher Scientific) supplemented with Halt protease and phosphatase inhibitors (Cat# 78446, Thermo Fisher Scientific). Cytoplasmic and nuclear fractions were prepared using a cytoplasmic and nuclear protein extraction kit (BRARZ106, BioTools Co. Ltd., Taiwan), following the manufacturer’s instructions. The concentration of proteins was determined using the Bradford method. The proteins were resolved using SDS-PAGE. The resolved proteins were transferred to a polyvinyl difluoride membrane. The membrane was incubated with the indicated primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies in Tris-buffered saline with 5% (w/v) milk or 1% (w/v) bovine serum albumin. The primary antibodies used in this study including anti-E-cadherin (ab238099; Abcam, Cambridge, MA, USA), anti-beta actin antibody (ab8227; Abcam, Cambridge, MA, USA), anti-active β-catenin (#8814; Cell Signal, Danvers, MA, USA), anti-p65 (ab16502; Abcam, Cambridge, MA, USA), and anti-TBP antibodies (ab28175; Abcam, Cambridge, MA, USA). Immunoreactive signals were visualized using an enhanced chemiluminescent detection reagent (GE Healthcare Life Sciences).