Superkine west femto maximum sensitivity substrate

Western blotting was performed to detect the expression of proteins related to cell adhesion. Total proteins were extracted from cells of each group using RIPA lysis buffer (Beyotime, Shanghai, China). A BCA protein assay kit (Beyotime, Shanghai, China) was used to measure the concentration of harvested proteins. An equal amount of 25 μg protein of each group was subjected to SDS-PAGE, followed by blotting onto a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA). After blocking with 10% skimmed milk, the membrane was probed overnight at 4°C using specific primary antibodies including integrin β1 (26918-1-AP,1:1000, ProteinTech, China), Fibronectin (15613-1-AP, 1:1000, ProteinTech, China), Integrin α6 (27189-1-AP, 1:1000, ProteinTech, China), and Integrin β4 (21738-1-AP, 1:1000, ProteinTech, China), followed by incubation with a horseradish peroxidase–conjugated secondary antibody, HRP-goat anti-mouse or HRP-goat anti-rabbit, for 1 hr at room temperature. The signal was visualized with a SuperKine™ West Femto Maximum Sensitivity Substrate (BMU102-CN, Abbkine) using Invitrogen iBright CL1500 imaging system (Thermo Fisher Scientific, USA). Bands data from Western blot were analyzed via iBright Analysis Software (5.2.2, Thermo Fisher Scientific, USA).