Sc 373800

Total proteins were extracted from clinical tissue samples and cancer cells by radio-immunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, USA) containing protease inhibitor (Biyuntian, Shanghai) for 30 minutes at 4°C as described previously [16 (link),17 (link)]. Vector and experimental group cells were collected, washed 3 times with ice-cold PBS and then protein was extracted using RIPA lysate buffer containing protease inhibitor (Biyuntian, Shanghai). Denatured proteins (50 ug) were separated using 12% SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and blotted onto nitrocellulose membranes (polyvinylidene difluoride). The membranes transferred by cell lysates were subsequently incubated with primary antibodies including: β-catenin (1: 1000; ab16051; Abcam, USA), GSK-3β(1: 1000; ab141295; Abcam), p-GSK-3β (1: 1000; sc-373800; Santa Cruz, USA), E-cadherin (1: 1000; ab197751; Abcam), N-cadherin (1: 1000; ab202030; Abcam), vimentin (1: 1000; ab92547; Abcam), OCT4n (1: 1000; ab181557; Abcam), and SOX2 (1: 1000; ab97959; Abcam). After 24 hours, the membranes were washed with PBS twice and then incubated with horseradish peroxidase-conjugated goat anti-rabbit for 2 hours at 37°C, immunoreactive bands were analyzed with by applying Imaging System (Thermo Fisher Scientific, USA). All assays were repeated thrice.