Anti forkhead box o3 foxo3

The total protein of cells and tissues was extracted using lysis buffer (Genechem, China) supplemented with protease inhibitor (Complete mini, USA). The protein concentration was measured using the bicinchonininc acid (BCA) protein assay kit (Beyotime Biotechnology, China).Then proteins were loaded and separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidenedifloride (PVDF) membranes. The membranes were subsequently blocked with 5% non-fat milk for 2 h and incubated with primary antibodies overnight at 4 °C. The primary antibodies used were as follows: anti-RAP1A (1:1000, Abcam, UK), anti-Forkhead box O3 (FOXO3) (1:1000, Cell Signaling Technology, USA), anti-PTEN (1:1000, Abcam), anti-cyclin D1 (CCND1, 1:1000, Abcam) and anti-β-actin (1:5000, Abcam) or anti-GAPDH (1:5000, Santa Cruz Biotechnology, USA). The membranes were incubated with secondary antibody (1:5000, Santa Cruz Biotechnology) for 1 h at room temperature. Finally, the blots were visualized using Chemiluminescence Detection Kit (Thermo Fisher Scientific). The protein expression was quantified using Quantity One software and β-actin or GAPDH served as the internal control.

res-TPC-1 and res-IHH-4 cells were harvested and lysed in RIPA buffer (Beyotime). Cell lysates were quantified by BCA kit (Thermo Fisher) and separated by SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked using specific blocking solution and then interacted with primary antibody anti-forkhead box O3 (FOXO3) (Cell Signaling Technology, Danvers, MA, USA) and the HRP-labeled secondary antibody IgG (Cell Signaling Technology). Anti-β-actin (Cell Signaling Technology) acted as a loading control. The ECL Western Blotting Substrate kit (Solarbio, Beijing, China) was used to develop the protein signals.