Ab76311

Total protein was extracted from the EC cell lines using a radioimmunoprecipitation assay (RIPA) kit (Solarbio). The protein samples were separated by 10% SDS–PAGE (Sangon Biotech, Shanghai, China) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, USA) according to a standard process. After blocking in 5% skim milk (BD Difco, Maryland, USA) or 5% bovine serum albumin(BSA, Biofroxx, Einhausen, Germany) for 2 h, the membranes were incubated overnight at 4 ^oC with primary antibodies against DCLK1 (JA11-03, HUABIO, Hangzhou, China), E-cadherin (ab40772, Abcam, Cambridge, UK), N-cadherin (ab76011, Abcam), Cyclin D1 (26,939-1-AP, Proteintech, Wuhan, China), CDK4 (#12,790, Cell Signaling Technology, Massachusetts, USA), PI3K p85 (ab191606, Abcam), p-PI3K p85 (phospho Y607) (ab182651, Abcam), Akt (60,203-2-lg, Proteintech), p-Akt (phospho Ser 473) (66,444-1-lg, Proteintech), NF-κB p65 (ab76311, Abcam), or NF-κB p-p65 (phospho S536) (ab76302, Abcam), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies(LF101 or LF102, Epizyme, Shanghai, China) for 2 h (1:5000). β-actin (AP0060, Bioworld, Nanjing, China) or GAPDH (AP0063, Bioworld) antibodies were used as the internal controls at a dilution of 1:5000. The protein signals were analyzed using the enhanced chemiluminescence method (Millipore).