Total p65

Cells and tissues were lysed using the Protein Extraction kit (Wanlei Biotechnology, Beijing, China). The proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PDVF) membranes. The membranes were blocked with skimmed milk (5%, w/v). The membranes were probed with anti-E-cadherin (1: 2000; Abcam, Cambridge, MA, USA), anti-Vimentin (1: 3000; Abcam, Cambridge, MA, USA), anti-NUS1 (1: 2000; Abcam, Cambridge, MA, USA), p-AKT (1: 5000; Abcam), total-AKT (1: 2000; Abcam), p-IKKα (1: 2000; Abcam), total-IKKα (1: 2000; Abcam), total-p65 (1: 3000; Abcam), p-p65 (1: 2000; Abcam), and anti-GAPDH (1: 3000; Abcam) antibodies overnight at 4˚C. The membranes were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h and the immunocomplexes were then visualized using a New Super ECL Detection Kit (KeyGEN BioTECH, China) according to the manufacturer’s protocol.

For Western blotting analysis, the cells were harvested and lysed on ice for 30 min in RIPA buffer supplemented with protease inhibitors (100 mM Tris–HCl at pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X‐100, 1% deoxycholate acid, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 2 mM DTT, 2 mM leupeptin, 2 mM pepstatin). Cells lysates were centrifuged at 12,000 rpm for 15 min, and the supernatants were collected as total proteins. After the concentrations of protein samples were determined by the BCA method (Beyotime, Haimen, China), an equal amount of each sample was separated by SDS‐PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% nonfat dried milk solution for 2 h and incubated with primary antibodies, respectively. The antibodies used were against cl‐caspase 3, Bcl2, Kras, Hif1α, PHD2 (CST), phospho‐P65, total P65 (Abcam, Cambridge, MA) and β‐actin (Boster Bio Tec, Wuhan, China). After washing three times with PBST, the membranes were incubated with HRP conjugated secondary antibody and visualized with an ECL detection system. Protein expression was measured by ImageJ software.