Jan 27, 2026

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

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l6e38rgqe/v1
  • Marvin De los Santos1
  • 1ChordexBio
Marvin De los Santos
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6e38rgqe/v1
Protocol Citation: Marvin De los Santos 2026. SEAP reporter assay for assessing NF-κB inhibition by TGF-β1/PD-L1 fusion protein. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6e38rgqe/v1
Manuscript citation:
De Los Santos, M. I., & Bernal, S. D. (2024). U.S. Patent No. 11,912,751. Washington, DC: U.S. Patent and Trademark Office. De Los Santos, Marvin I. (2019). Construction and Characterization of a Novel Bifunctional TGF-B1/PD-L1 Fusion Protein (M.S. in Miolecular Biology and Biotechnology Thesis). University of the Philippines Diliman. College of Science. National Institute of Molecular Biology and Biotechnology. UP Diliman CS Library. https://cslib.science.upd.edu.ph/
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 26, 2026
Last Modified: January 27, 2026
Protocol  Integer ID: 241572
Keywords: NF-κB signaling, SEAP reporter assay, secreted alkaline phosphatase, secreted embryonic alkaline phosphatase, HEK-Blue TLR2 cells, PD-1 receptor, TGF-β1/PD-L1 fusion protein, Immunomodulation, Inflammatory signaling, Reporter gene assay, κb inhibition by tgf, κb activation, κb pathway inhibition, κb inhibition, κb, l1 fusion protein this protocol, l1 fusion protein, seap reporter assay, tlr2 reporter hek 293 cell, human tlr2 reporter hek 293 cell, immunomodulatory protein, signaling pathway, treatment with the tgf, dependent inhibition of nf, seap secretion into the culture medium, tgf
Abstract
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.
Protocol materials
Imject™ Freund's Complete Adjuvant (FCA)Thermo FisherCatalog #77140
HEK Blue Detection MediumInvivoGen
Safety warnings
This assay is sensitive to the presence of endotoxins. Before performing the experiment, ensure first that your culture is not contaminated and your reagents are endotoxin-free.
Before start
This protocol uses Human TLR2 Reporter HEK 293 Cells from InvivoGen, lipofected stably with pCMV3-PD1 to express the PD-1 receptor. Please contact the author regarding inquiries on how to reconstitute the PD-1 protein in these cells or different eukaryotic cell models.
Confirmation of receptor expression in PD1+ TLR2+ HEK 293 cells
44m
Perform immunofluorescence (IF) analysis using anti-TGFBR1 and anti-PD1 antibodies. Note that you can use FITC-conjugated antibodies (direct) or premix the primary antibodies with FITC-conjugated secondary antibodies in 1:1 ratio (indirect)
Harvest 1 x106 cells and collect by centrifugation 500 x g, 25°C, 00:05:00
5m
Resuspend the cell pellet in 100 µL of PBS, repeat once
Add 100 µL of staining buffer and resuspend the cells well by pipetting
AB
ReagentAmount needed
Bovine serum albumin (BSA)0.05% (w/v)
FITC-labelled antibody1:100 (antibody:buffer ratio)
PBSFill to 100 uL
Composition of staining buffer

Incubated at 4 °C for 00:30:00 in the dark with constant shaking. Note that over-exposure to light may photobleach FITC signal

30m
Wash the cells in 5 mL PBS-T, twice.
AB
ReagentAmount needed
Tween-200.01% (w/v)
PBS5 mL
Composition of PBS-T buffer

Collect the cells by centrifugation 500 x g, 25°C, 00:03:00 and resuspend the cells in 25 µL of PBS with or without 0.5 μg/mL of Hoechst 33342. Incubate for 00:05:00 at room temperature
8m
Wash the cells once with 5 mL PBS and resuspend to a final volume 100 µL of PBS
Aliquot desired amount and dispense to poly-L-lysine coated slides, incubate in the dark for at least 00:01:00 prior to visualization under a fluorescence microscope
1m
SEAP reporter assay
2d
Seed 1x105 cells in a 96-well plate, each well should not exceed 200 µL
After 24:00:00 , replace the medium with same volume of HEK Blue Detection MediumInvivoGen
1d
Challenge the cells with an increasing dilution of Mycobacteria-containing Imject™ Freund's Complete Adjuvant (FCA)Thermo FisherCatalog #77140

AB
Set-upDilution
11:100
21:1,000
31:10,000
41:50,000
51:100,000
ControlNone (PBS only)
Dilution reactions for the experiment

Note: Consider the number of replicates in your assays
Divide the set-up into fusion protein-treated, antibody-blocked, and untreated reactions. For this protocol, the inhibition of NF-κB activation by the TGF-β1/PD-L1 (TP) fusion protein is determined. Note that you can use a different immunomodulating protein or molecule for this assay.
AB
ReactionPurpose
TP-treatedAssess the direct effect of receptor binding of TP with receptors in NF-κB activation
Antibody-blockedIdentify the consequences of blocking the receptors of TP in NF-κB activation
UntreatedServes as control
SEAP reporter assay set-up; TP = TGF-β1/PD-L1 fusion protein

For TP-treated cells, add 50 ng/mL of fusion protein to the wells
For antibody-blocked cell, add 1 ug/mL of either anti-TGFBR1 and anti-PD1 antibodies, incubate for 00:15:00 , then add 50 ng/mL of fusion protein to the wells

For untreated cells, add the same volume of PBS to the wells
Culture the cells as usual for 24:00:00

1d
Check the culture plate for color development, image the plate and then read the optical density (OD) at 620-655 nm on a plate reader