In Situ Protein-Protein Interaction Assay

Interactions between two molecules were determined using in situ PLA (Duolink^® In Situ reagents, O-LINK^® Bioscience, Sweden) as described previously (Lee et al., 2016 (link)). Briefly, cells were plated onto round coverslips in 24-well cell culture plates and grown for 24 h in complete DMEM. The cells were serum-starved for 6 h in DMEM with 0.1% BSA, then pretreated with each reagent as indicated (TNFR2-SKE for 0.5 h, DEXamethasone (DEX; Sigma, USA) for 3 h, or dehydroascorbic acid (DHA; Sigma, USA) for 1 h at 37°C in a 5% CO₂ incubator). The cells were additionally stimulated with TNF-α (25 or 50 ng/ml), followed by washing twice with 1× PBS. Cells were fixed with 2% formaldehyde in PBS for 15 min at room temperature. All procedures for in situ PLA were performed according to the manufacturer’s recommended protocol and protein-protein interactions were analyzed using a confocal laser-scanning microscope (Olympus FluoView FV1000, Olympus, Japan). We used antibodies against the following target proteins: TNFR1, TRAF2, NF-κB/p50 (Santa Cruz Biotechnology), and NF-κB/p65 (Cell Signaling Technology).