Ab110644

Cells were inoculated in a 96-well plate at a concentration of 1x10³/well. The cells were treated with LPS, as previously described, and then total protein was extracted from the cells using RIPA buffer (Auragene; Hunan Aijia Biotechnology Co., Ltd.). Protein quantities were determined using a BCA protein assay kit (Bio-Rad Laboratories, Inc.). Samples containing 40 µg protein/lane were separated by 10% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). Following blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies targeting TRADD (1:1,000; cat. no. ab110644; Abcam), p65 (1:1,000; cat. no. ab16502; Abcam), phosphorylated (p)-p65 (1:1,000; cat. no. ab183559; Abcam) and GAPDH (1:1,000; cat. no. ab9485; Abcam) overnight at 4˚C. Following washing with 0.1% PBS-Tween-20 four times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000; ab150113; Abcam) for 1 h at room temperature. Finally, the bands were visualized using an ECL detection system (Beyotime Institute of Biotechnology) and Image J software (version 146; National Institutes of Health) was used to analyze the fold-changes of protein levels.

Paraffin sections were fully deparaffinized and hydrated, and then washed and heated to 92-98 °C for antigen retrieval. Samples were incubated with primary antibodies against A20 (ab13597, Abcam), RIP1 (ab72139, Abcam), and TRADD (ab110644, Abcam) in blocking buffer overnight at 4 °C. Samples were subsequently incubated with the corresponding secondary antibody in blocking buffer at room temperature, and finally incubated with DAPI staining solution for 10 min. Images were obtained under a fluorescence microscope (Nikon, Japan).