Anti foxo1

Western blotting analyses were performed as previously described using 30μg lysates from fresh tissues and cells [6 (link)]. Proteins were visualized by exposing Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) by protein imprinting imaging system (Tanon5200, China). Antibodies against human GDF15, AKT1, Erk1/2, GAPDH were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-PI3K (p-p110a) was from Abcam Technology (Cambridge, MA, USA). Anti-p-AKT was purchased from CST (Littleton, CO, USA). Anti-Ras and Anti-Ras-GTP was from NewEast Biosciences (WuHan, CN). Anti-p-Erk1/2, anti-ErbB2, anti-p-ErbB2, anti-FOXO1 were from Abclonal Technology (Boston, MA, USA).

Total protein of cells was extracted through ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, 89,900), containing the protease inhibitor cocktail (Merk, P8340). Then, the proteins were quantified by BCA Protein Assay Kit (Beyotime, P0010) and boiled for degeneration. Subsequently, we separated proteins in SDS-PAGE (Beyotime, P0012A) and transferred them on the PVDF membrane (Merk, 3,010,040,001). After being blocked in 5% Bovine serum albumin (BSA, Solarbio, SW3015), membranes were incubated with primary antibodies: Anti-FOXO1 (Abclonal, A2934, 1:1000) and Anti-beta-actin (Proteintech, 20,536, 1:1000). Then, the membranes were incubated in secondary antibodies: Goat Anti-Mouse IgG (Proteintech, SA00001-1, 1:1000) and Goat Anti-Rabbit IgG (Proteintech, SA00001-2, 1:1000), followed by enhanced chemiluminescence to display bands.