Anti ndfip2

The cells were washed with PBS, lysed with radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) containing 1% 10 µM Phenylmethanesulfonyl uoride (PMSF) and 1% phosphatase inhibitor. Quantitative protein concentration using bicinchoninic acid kit (BCA, Vazyme Biotechnology Co., Ltd., Nanjing, China). After protein reduction and denaturation, the equal amount of protein lysis and Page Ruler prestained protein ladder were loaded into 10% SDS-PAGE gel, then transferred to polyvinylidene uoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5 % bovine serum albumin (BSA) and incubated (overnight, 4°C) with primary antibodies. Western blot was performed using antibodies that anti-NDFIP2(1:1000, Bioss antibodies Inc., Beijing, China. Cat # bs-19059R), anti-pan AKT (1:1000, Abcam, Cambridge, UK, Cat # ab8805), anti-AKT1 phospho (1:1000, Abcam, Cat # ab66138), anti-N cadherin (1:1000, Abcam, Cat # ab18203), anti-E cadherin (1:10000, Abcam, Cat # ab40772). After washings with TBS-T, membranes were incubated with the secondary antibody for 1 hour at room temperature. Protein bands are visualized with Western chemiluminescent HRP substrate (Millipore, Billerica, MA, USA). Anti-β-actin antibody (1:2000, Sigma, USA) is used as an internal control to ensure the equal amount of protein loading.