Trim28

Tumor tissue or cells were homogenized and lysed in RIPA buffer supplemented with a protease inhibitor cocktail and Phenylmethylsulfonyl Fluoride (Sigma). Protein samples were separated by SDS-PAGE and transferred onto PVDF membranes (Bio-Rad). Monoclonal/polyclonal primary antibodies and appropriate HRP-conjugated secondary antibodies were employed for Western blotting. Protein visualization was achieved using ECL-Plus. For immunoprecipitation (IP), cells were lysed, and protein concentrations were quantified. Primary antibodies were added and incubated overnight at 4 °C with rotation. Protein G beads were added and incubated for 3 h at 4 °C with rotation. The resin was centrifuged, washed three times with lysis buffer, and analyzed by immunoblotting.
The following antibodies were used: TRIM28 (Cat: PA5-27648, Invitrogen), RIPK1 (Cat: PA5-20811, Invitrogen), Phospho-IκBα (Cat: #2859, Cell Signaling Technology), IκBα (Cat: #4814, Cell Signaling Technology), Phospho-IKKα/β (Cat: #2697, Cell Signaling Technology), IKKα (Cat: #2682, Cell Signaling Technology), IKKβ (Cat: #8943, Cell Signaling Technology), anti-Flag (Cat: #14,793, Cell Signaling Technology), anti-Ubiquitin (Cat: #3936, Cell Signaling Technology), anti-K48-linkage Specific Polyubiquitin (Cat: #8081, Cell Signaling Technology), and anti-K63-linkage Specific Polyubiquitin (Cat: #5621, Cell Signaling Technology).

Immunohistochemistry staining was performed according to the manufacturer’s instructions. Briefly, tissue slides were deparaffinized, rehydrated, and treated with 3% H₂O₂ solution for 15 min at room temperature to block endogenous peroxidase activity. Slides were then subjected to antigen retrieval in EDTA buffer (pH 9.0) at 96 °C for 20 min. After blocking with 10% goat serum, slides were incubated with primary antibodies at 4 °C overnight. The following antibodies were used: TRIM28 (Cat: PA5-27648, Invitrogen), CXCL1(Cat: PA5-86508, Invitrogen), anti-CD8(Cat: ab237723, abcam), and anti-S100A8 + S100A9(Cat: ab288715, abcam). Following incubation with secondary antibodies and development with 3,3-diaminobenzidine, the final IHC score was calculated based on staining extent and intensity. Staining extent was scored based on the percentage of positively stained cells (0 to 3), while staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). Specimens with final scores ≥ 2 were considered to have high expression, while those with final scores < 2 were considered to have low expression. For immune cell markers where signal intensity was not considered, the percent positive cell score was calculated based on the percentage of positively stained cells.