Calvarial osteoblasts or U2OS cells were plated at a density of 50% in 12-well plates in replicates of 6. As indicated, cells were transfected with 250 ng of the TOP FLASH reporter and/or various expression vectors (empty pcDNA4.0, Flag-tagged TIEG1, Flag-tagged Lef1, constitutively active β-catenin or Xpress-tagged TIEG1 domain expression constructs (35 (link))) using Fugene-6 (Roche, Indianapolis, IN, USA) as specified by the manufacturer. Empty vector was added to transfections as necessary to normalize the total amount of DNA transfected across each condition. Twenty four hours following transfection, cells were lysed in passive lysis buffer (Promega, Madison, WI), lysates were quantitated for protein content and equal amounts of protein were used to measure luciferase activity using Luciferase Assay Reagent (Promega) and a Glomax-Dual luminometer (Promega).
Protocol detail
Luciferase Assay of Osteoblast Transfections
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Assay
Buffer
Calvarial
Cells
Fugene 6
Lef1
Luciferase
Osteoblasts
Promega
Protein
Transfection
Vector
β catenin
Corresponding Organization : WinnMed
Other organizations : Most Sacred Heart of Jesus Cardiology and Valvular Institute, Mayo Clinic in Florida, Baylor University
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Variable analysis
independent variables
- Transfection with 250 ng of the TOP FLASH reporter
- Transfection with various expression vectors (empty pcDNA4.0, Flag-tagged TIEG1, Flag-tagged Lef1, constitutively active β-catenin, or Xpress-tagged TIEG1 domain expression constructs)
- Luciferase activity
- Calvarial osteoblasts or U2OS cells were plated at a density of 50% in 12-well plates in replicates of 6
- Empty vector was added to transfections as necessary to normalize the total amount of DNA transfected across each condition
- Twenty four hours following transfection, cells were lysed in passive lysis buffer (Promega, Madison, WI), lysates were quantitated for protein content and equal amounts of protein were used to measure luciferase activity using Luciferase Assay Reagent (Promega) and a Glomax-Dual luminometer (Promega)
- Empty pcDNA4.0 vector
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