The luciferase reporter assay was performed as described previously (2 (link)). In short, 4D6 cells were cotransfected with a renilla control plasmid (pRL Promega) and a luciferase reporter plasmid (pGL4.10[Luc2], Promega) under the control of a minimal wild-type Psmb11 promoter (designated β5t-luc) or a β5t promoter with a mutated FOXN1-binding sites (β5t-mut-luc) plus a FOXN1 construct of interest in a ratio of 1:10:10. Luciferease activity was measured 24 hours later using the Dual-Lucifearase Assay kit (Promega).
Rota I.A., Handel A.E., Maio S., Klein F., Dhalla F., Deadman M.E., Cheuk S., Newman J.A., Michaels Y.S., Zuklys S., Prevot N., Hublitz P., Charles P.D., Gkazi A.S., Adamopoulou E., Qasim W., Davies E.G., Hanson I., Pagnamenta A.T., Camps C., Dreau H.M., White A., James K., Fischer R., Gileadi O., Taylor J.C., Fulga T., Lagerholm B.C., Anderson G., Sezgin E, & Holländer G.A. (2021). FOXN1 forms higher-order nuclear condensates displaced by mutations causing immunodeficiency. Science Advances, 7(49), eabj9247.
Other organizations :
Great Ormond Street Hospital, University College London, Baylor College of Medicine, Wellcome Centre for Human Genetics, National Institute for Health Research, University of Birmingham, MRC Human Immunology Unit
Type of luciferase reporter plasmid (wild-type Psmb11 promoter (β5t-luc) or mutated FOXN1-binding sites (β5t-mut-luc))
Presence of a FOXN1 construct of interest
dependent variables
Luciferase activity
control variables
Cotransfection with a renilla control plasmid (pRL Promega)
Cell line (4D6 cells)
controls
Positive control: Luciferase reporter plasmid with wild-type Psmb11 promoter (β5t-luc)
Negative control: Luciferase reporter plasmid with mutated FOXN1-binding sites (β5t-mut-luc)
Annotations
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