Transcriptional Regulation of IL-10 in HEK293T Cells

10⁴ HEK293T cells were transfected with IL-10 firefly luciferase reporter constructs, renilla luciferase reporter and plasmids expressing IRF1, BATF, W11R^{30 (link)} or c-Maf using Fugene. Cells were analyzed 48h later with the dual luciferase assay kit (New England Biolabs). Fragments containing the proximal Il10 promoter (−1.5 kb including the HSS-0.12 site), and the CNS-9 or HSS⁺2.98 regions upstream of the Il10 minimal promoter were described before ^{9 (link), 31 (link)} and were cloned into pGL4.10 Luciferase reporter plasmid (Promega).

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Karwacz K., Miraldi E.R., Pokrovskii M., Madi A., Yosef N., Wortman I., Chen X., Watters A., Carriero N., Awashti A., Regev A., Bonneau R., Littman D, & Kuchroo V.K. (2017). Critical role of the transcription factors IRF1 and BATF in preparing the chromatin landscape during Type 1 regulatory cell differentiation. Nature immunology, 18(4), 412-421.

Publication 2017

pGL4.10 Luciferase reporter plasmid

Assay Cells Firefly luciferase Fugene Il10 Irf1 Luciferase Pgl4 Plasmid Promega Renilla luciferase

Corresponding Organization : Harvard University

Other organizations : New York University, Simons Foundation, University of California, Berkeley, Broad Institute, Massachusetts Institute of Technology, Translational Health Science and Technology Institute

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Variable analysis

independent variables

Expression of IRF1
Expression of BATF
Expression of W11R
Expression of c-Maf

dependent variables

Firefly luciferase reporter activity
Renilla luciferase reporter activity

control variables

HEK293T cell line
Transfection using Fugene
Dual luciferase assay kit (New England Biolabs)
Il10 promoter and upstream regulatory regions (CNS-9, HSS+2.98, HSS-0.12) cloned into pGL4.10 Luciferase reporter plasmid

controls

Positive control: Cells transfected with IL-10 firefly luciferase reporter constructs, renilla luciferase reporter, and plasmids expressing transcription factors (IRF1, BATF, W11R, c-Maf)
Negative control: Not explicitly mentioned

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