Detection of TNF-α levels in supernatant via ELISA
Supernatant was investigated for levels of TNF-α protein using commercial TNF-α ELISAs (Hengyuan Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s instructions.
Western blot
Cells were harvested, and total protein lysates were resolved using 10% sodium dodecyl sulfate–polyacrylamide gels,electroblotted onto a polyvinylidene fluoride membrane,blocked by 5% skim milk, and probed with primary antibodies listed in Table S1. The membrane was then incubated with the secondary antibody and visualized by enhanced chemiluminescence using Kodak X-Omat LS film (Eastman Kodak,Rochester, USA).
