Western Blot and Cytokine Analysis of Macrophage Responses

The total protein from RAW264.7 macrophages was obtained by using the RIPA buffer (Beyotime). A nuclear and cytoplasmic extraction kit (CWbiotech) was used to, respectively, obtain the cytosolic protein and nuclear protein. A total of 30 μg protein in each lane was prepared, followed by separating in a 10% polyacrylamide SDS-PAGE gel [30 (link)]. After transferring, the PVDF membranes were then blocked with 5% BSA, then incubated with different antibodies including NRF2, Histone H3, p-JNK, KEAP1, DRP1, p-ERK, FIS1, ZBTB20, p-p65, IκBα, p-p38, IRF3, p-IRF3, and GAPDH (Cell Signaling Technology) [31 (link)]. After the incubation, a secondary antibody with linked HRP (Cell Signaling Technology), an ECL detection kit (Yeasen Biotech Co., Ltd.), and a digital imaging system (Kodak) were used.
To detect the cytokines, including TNF-α, IL-6, and IFN-β, the macrophage supernatants were collected, followed by detection with ELISA kits (Neobioscience Technology Co., Ltd.).