LILRB3 Signaling Activation Assay

THP-1-Lucia^™ NF-κB cells were purchased from InvivoGen. Human anti-LILRB3 antibody with the N297A mutation was coated onto the plate to activate LILRB3 signaling, and plates coated with hIgG (N297A) were used as the control. The activation of NF-κB signaling was evaluated by monitoring luciferase signal. Infected THP-1 reporter cells were cultured for an additional month before stimulation with anti-LILRB3. For the NF-κB reporter assay conducted in 293T cells, an NF-κB-driven firefly luciferase reporter plasmid co-transfected with a plasmid encoding CMV-driven Renilla luciferase along with plasmids expressing LILRB3, TRAF2, or cFLIP were transfected into cells. The luciferase activity was detected using the Dual-Luciferase® Reporter (DLR^™) Assay System (Promega). LILRB3 chimeric receptor reporter cells were constructed as we described^{68 (link)–70 (link)}, with LILRB3-ECD fused with the transmembrane and intracellular domains of paired immunoglobulin-like receptor β, which signals through the adaptor DAP-12 to activate the NFAT promoter.