cDNA clone of LRP6 (pCR-XL-TOPO-LRP6) was purchased from FulenGen. cDNA sequence was validated by sequencing and inserted into pShuttle-IRES-hrGFP-1 vector. Mutations (Y418H and R611C, a previously reported mutation [12 (link)]) were introduced by PCR-based mutagenesis, respectively. The mutations were verified by DNA sequencing. HUVEC was electric transfected with equal amount of plasmids expressing wild-type (LRP6wt) and mutant LRP6 (LRP6R611C or LRP6Y418H), respectively. A Wnt pathway reporter system was utilized as previously described [12 (link)]. Briefly, plasmids encoding wild-type LRP6 or mutants, LEF-1, firefly luciferase, and renilla luciferase were introduced into cells by transfection. After 24 h, cells were lysed and luciferase reporter activity indicating the extent of Wnt signal activation was measured in accordance with the dual luciferase assay specifications (Promega, Hollow Road Madison, WI, USA).
Dual luciferase assay specifications
Manufactured by Promega
Sourced in United States, Germany
The Dual Luciferase Assay is a laboratory tool used to measure the activity of two luciferase reporter genes simultaneously. It provides a rapid and sensitive method for quantifying gene expression levels. The assay utilizes two different luciferase enzymes, Firefly and Renilla, which emit light at different wavelengths upon the addition of their respective substrates. This allows for the normalization of experimental results, improving the accuracy of gene expression analysis.
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Lab products found in correlation
2 protocols using dual luciferase assay specifications
1
LRP6 Mutation Effects on Wnt Signaling
2
Dual-Luciferase Reporter Assay for Transcriptional Activity
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Cells were seeded on a 24-well plate, and transfected with siRNAs. After 48 h, cells were transfected (GeneCopoeia) with 500 ng TOP flash or NF-κB reporter and 10 ng pRL-TK (Promega, Madison, WI, USA) plasmids using EndoFectin™-Plus. Assays were performed in accordance with the dual-luciferase assay specifications (Promega) using the Mithras LB 940 luminometer (Berthold, Bad Wildbad, Germany). The activity of firefly luciferase was normalized to measure the transfection efficiency. All experiments were performed at least 3 times.
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