SEAP reporter assay for assessing NF-κB inhibition by TGF-β1/PD-L1 fusion protein v1

This protocol describes a secreted embryonic alkaline phosphatase (SEAP)–based reporter assay to evaluate NF-κB pathway inhibition mediated by a TGF-β1/PD-L1 fusion protein. Human TLR2 Reporter HEK 293 cells are stably modified to express the PD-1 receptor and used as a cellular model to assess downstream NF-κB activation following Toll-like receptor 2 stimulation. NF-κB signaling is induced using graded dilutions of Mycobacteria-containing Freund’s Complete Adjuvant, and pathway activity is quantified via SEAP secretion into the culture medium. Treatment with the TGF-β1/PD-L1 fusion protein is compared against antibody-blocked and untreated controls to determine receptor-dependent inhibition of NF-κB activation. This assay provides a reproducible and modular framework for mechanistic validation of immunomodulatory proteins affecting inflammatory signaling pathways.