Ckmt1 Promoter Activation Assay in Macrophages

The Ckmt1 promoter reporter construct has been described previously (Fenouille et al, 2017 (link)). Mouse macrophage cell line RAW 264.7 (ATCC, TIB‐71) was cotransfected with the Ckmt1 promoter construct expressing Gaussia Luciferase and Secreted Alkaline Phosphatase (GeneCopoeia) and the Zeb1 plasmid using GenJet DNA Transfection Reagent (SignaGen Laboratories) in accordance with the manufacturer's protocol. Transfection medium was replaced by fresh medium containing RANKL about 6 h after transfection. After 48 h of incubation, medium was collected for parallel bioluminescence assays of Gaussia luciferase and secreted alkaline phosphatase using Secrete‐Pair Dual Luminescence Assay Kit (Genecopoiea), using a SpectraMax L (Molecular Devices) plate reader.

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Zhu L., Tang Y., Li X., Kerk S.A., Lyssiotis C.A., Feng W., Sun X., Hespe G.E., Wang Z., Stemmler M.P., Brabletz S., Brabletz T., Keller E.T., Ma J., Cho J., Yang J, & Weiss S.J. (2023). A Zeb1/MtCK1 metabolic axis controls osteoclast activation and skeletal remodeling. The EMBO Journal, 42(7), e111148.

Publication 2023

GenJet DNA Transfection Reagent SpectraMax L

Alkaline phosphatase Bioluminescence assays Cell line Devices Luciferase Luminescence assay Macrophage Mouse Plasmid Rankl Transfection

Corresponding Organization :

Other organizations : University of Michigan–Ann Arbor, Wuhan University, Friedrich-Alexander-Universität Erlangen-Nürnberg

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Variable analysis

independent variables

Zeb1 plasmid

dependent variables

Gaussia Luciferase activity
Secreted Alkaline Phosphatase activity

control variables

Mouse macrophage cell line RAW 264.7
GenJet DNA Transfection Reagent
RANKL treatment

controls

Ckmt1 promoter reporter construct expressing Gaussia Luciferase and Secreted Alkaline Phosphatase

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