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Dual specific luciferase reporter assay system

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The Dual-specific luciferase reporter assay system is a laboratory tool designed to measure the activity of two different luciferase reporter genes simultaneously within the same sample. It provides a quantitative method for analyzing gene expression or monitoring cellular signaling pathways.

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Lab products found in correlation

Nf κb, by Promega (2 mentions) Ap 1 reporter plasmid, by Promega (2 mentions) Syk inhibitor r406, by InvivoGen (2 mentions) Passive lysis buffer (plb), by Promega (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Prl tk renilla luciferase reporter plasmid, by Promega (1 mentions) Lys pgn, by Merck Group (1 mentions) Fugene hd transfection reagent, by Roche (1 mentions)

5 protocols using dual specific luciferase reporter assay system

1

Investigating NF-κB and AP-1 Signaling

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HEK293 cells were transfected with 200 ng of expression plasmid, and 10 ng NF-κB or 100 ng AP-1 reporter plasmid (Promega). To normalize the transfection efficiency, 10 ng pRL-TK Renilla Luciferase reporter plasmid was used. After 18 hours transfection, cells were stimulated with 20 ng/mL TNF or phorbol myristate acetate (PMA) (InvivoGen), and 2 μM SYK inhibitor R406 (InvivoGen) for 16 hours before measurement. Cells were harvested using passive lysis buffer (Promega) and Luciferase assays were carried out using a dual-specific Luciferase reporter assay system (Promega) according to the instructions. Each experiment was performed in triplicate.
Wang L., Aschenbrenner D., Zeng Z., Cao X., Mayr D., Mehta M., Capitani M., Warner N., Pan J., Wang L., Li Q., Zuo T., Cohen-Kedar S., Lu J., Ardy R.C., Mulder D.J., Dissanayake D., Peng K., Huang Z., Li X., Wang Y., Wang X., Li S., Bullers S., Gammage A.N., Warnatz K., Schiefer A.I., Krivan G., Goda V., Kahr W.H., Lemaire M., Lu C.Y., Siddiqui I., Surette M.G., Kotlarz D., Engelhardt K.R., Griffin H.R., Rottapel R., Decaluwe H., Laxer R.M., Proietti M., Hambleton S., Elcombe S., Guo C.H., Grimbacher B., Dotan I., Ng S.C., Freeman S.A., Snapper S.B., Klein C., Boztug K., Huang Y., Li D., Uhlig H.H, & Muise A.M. (2021). Gain-of-function variants in SYK cause immune dysregulation and systemic inflammation in humans and mice. Nature genetics, 53(4), 500-510.
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2

Luciferase Reporter Assay for IFNλ

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293T cells (2×105 cells per well) were seeded into 24-well plates. The cells were transfected with 50 ng of luciferase reporter plasmids (a gift of Prof. Xiao Shaobo from HuaZhong Agricultural University) [32 (link)], together with pCAGGS-IFNλ or pCAGGS, using Lipofectamine 2000 (Thermo Scientific, Shanghai, China). In parallel, 20 ng of pRL-TK Renilla luciferase reporter plasmid (Promega, Madison, WI, USA) was transfected to normalize transfection efficiency. Twenty-four hours after transfection, the cells were infected with B2c, rB2c-IFNλ2, and rB2c-IFNλ3 for 12 h. Luciferase activity in total cell lysates was measured using a dual-specific luciferase reporter assay system (Promega, Madison, WI, USA).
Li Y., Zhao L., Luo Z., Zhang Y., Lv L., Zhao J., Sui B., Huang F., Cui M., Fu Z.F, & Zhou M. (2020). Interferon-λ Attenuates Rabies Virus Infection by Inducing Interferon-Stimulated Genes and Alleviating Neurological Inflammation. Viruses, 12(4), 405.
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3

Transfection and Luciferase Assay in Drosophila and HEK Cells

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DNA or RNA transfection was performed as previously described [47 (link)]. In brief, Drosophila S2 cells were plated in six-well plates with about 1 × 106 cells per well and then transfected with 2 μg of plasmids or RNAs using FuGENE HD Transfection Reagent (Roche) according to the manufacturer’s protocol. For PGN challenge, the cells were challenged by Lys-PGN [extract from S. aureus (Sigma)] 48 h after the transfection.
HEK293T or 293-TLR3 cells were plated on 24-well plates (2 × 105 cells per well) and transfected with the luciferase reporter plasmid (50 ng) together with the expression plasmid or empty control plasmid (0.5 μg each). For each transfection, we added 20 ng of pRL-TK Renilla luciferase reporter plasmid to normalize the transfection efficiency. In addition, empty vector was added to ensure that each transfection receives the same amount of total DNA. Luciferase activity in total cell lysates was measured with a dual-specific luciferase reporter assay system (Promega).
Wu D., Wang Z., Zhang J., Robinson A.G., Lyu B., Chen Z., Wang C., Wei B., Xia X., Zhang Q, & Zhou X. (2022). Apoptotic caspase inhibits innate immune signaling by cleaving NF-κBs in both Mammals and Flies. Cell Death & Disease, 13(8), 731.
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4

Investigating NF-κB and AP-1 Signaling

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HEK293 cells were transfected with 200 ng of expression plasmid, and 10 ng NF-κB or 100 ng AP-1 reporter plasmid (Promega). To normalize the transfection efficiency, 10 ng pRL-TK Renilla Luciferase reporter plasmid was used. After 18 hours transfection, cells were stimulated with 20 ng/mL TNF or phorbol myristate acetate (PMA) (InvivoGen), and 2 μM SYK inhibitor R406 (InvivoGen) for 16 hours before measurement. Cells were harvested using passive lysis buffer (Promega) and Luciferase assays were carried out using a dual-specific Luciferase reporter assay system (Promega) according to the instructions. Each experiment was performed in triplicate.
Wang L., Aschenbrenner D., Zeng Z., Cao X., Mayr D., Mehta M., Capitani M., Warner N., Pan J., Wang L., Li Q., Zuo T., Cohen-Kedar S., Lu J., Ardy R.C., Mulder D.J., Dissanayake D., Peng K., Huang Z., Li X., Wang Y., Wang X., Li S., Bullers S., Gammage A.N., Warnatz K., Schiefer A.I., Krivan G., Goda V., Kahr W.H., Lemaire M., Lu C.Y., Siddiqui I., Surette M.G., Kotlarz D., Engelhardt K.R., Griffin H.R., Rottapel R., Decaluwe H., Laxer R.M., Proietti M., Hambleton S., Elcombe S., Guo C.H., Grimbacher B., Dotan I., Ng S.C., Freeman S.A., Snapper S.B., Klein C., Boztug K., Huang Y., Li D., Uhlig H.H, & Muise A.M. (2021). Gain-of-function variants in SYK cause immune dysregulation and systemic inflammation in humans and mice. Nature genetics, 53(4), 500-510.
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5

TGF-β and VEGF Signaling Assays

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To assess TGF-β signaling, HEK293 cells or HEK293-hCD4 cells transfected with a TGF-β/SMAD Firefly luciferase reporter plasmid38 and a pRL-TK Renilla luciferase reporter plasmid were plated in 24-well plates at 2x105 cells per well in 500 μL of DMEM medium, and cultured for 18 hr at 37°C. Plates were subsequently incubated with serial dilutions of 4T-Trap or control antibodies in DMEM medium for 30 min, which were left unwashed or washed and re-cultured with 10 ng/mL recombinant human TGF-β1 in DMEM medium for 12 hr. Cells were subsequently lysed, and assyaed for luciferase activities with a dual-specific luciferase reporter assay system (Promega). To validate VEGF-Trap inhibition of VEGF signaling, HEK293 cells were co-transfected with a VEGF-responsive NFAT Firefly luciferase reporter plasmid39 , a VEGFR2 expression plasmid and a pRL-TK Renilla luciferase reporter plasmid. Plates were subsequently incubated with serial dilutions of VEGF-Trap and 10 ng/mL recombinant mouse VEGF165 (Cat. # 450-32, Peprotech) for 12 hr before luciferase activities were measured.
Li S., Liu M., Do M.H., Chou C., Stamatiades E.G., Nixon B.G., Shi W., Zhang X., Li P., Gao S., Capistrano K.J., Xu H., Cheung N.K, & Li M.O. (2020). Cancer Immunotherapy via Targeted TGF-β Signaling Blockade in TH Cells. Nature, 587(7832), 121-125.
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