K1034g af594

Total proteins were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, R0030, Beijing, China) and the protein concentration was quantified using a bicinchoninic acid (BCA) assay kit (Solarbio, PC0020, Beijing, China). A total of 20 μg protein was separated using 12% polyacrylamide gel and separated protein samples were transferred to polyvinylidene fluoride membranes (Millipore, IPVH00010, MA, USA). The membranes were blocked using 5% non-fat milk at ambient temperature for 1 h and incubated with primary antibodies against DR6 (Sigma-Aldrich, SAB1407346, MO, USA), p-AKT (CST, 4060, MA, USA), AKT (Abcam, ab38449, Cambridge, UK), p-NF-κB (CST, 3033, MA, USA), NF-κB ( ABclonal, 4790, Wuhan, China), and GAPDH (CST, 5174, MA, USA) at 4 °C for 12 h. After washing, the membranes were further labeled with goat anti-rabbit IgG secondary antibody (Solarbio, K1034G-AF594, Beijing, China) for 1 h at ambient temperature. GAPDH was used as the loading control. An enhanced chemiluminescence (ECL) kit (Beyotime, P0018M, Beijing, China) was used to visualize protein banes on the membrane. The grayscale values of each group of protein bands were determined using Image J software (NIH, Bethesda, MA, USA).