The 2 Kb mouse SOST promoter luciferase reporter vector used in this study was a kind gift from Dr. de Crombrugghe (MD Anderson) and its development was described previously (Zhou et al., 2010 (link)). Promoter deletion constructs (-1.95Kb and -1.8Kb) were generated by PCR amplification using the 2 Kb promoter construct as a template and subsequently cloned into the pGL3-basic luciferase reporter construct. The sequence integrity of all constructs was confirmed by Sanger sequencing. SOST promoter reporter constructs were transiently transfected into U2OS osteosarcoma cells using Fugene-6 (Roche, Indianapolis, IN) with either an empty pcDNA4-TO expression vector or a pcDNA4-TO mouse flag-tagged TIEG expression vector as previously described(Subramaniam et al., 2016 (link)). 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) (Hawse et al., 2011 (link); Hawse et al., 2008c (link)).
Glomax dual luminometer
Manufactured by Promega
The Glomax-Dual Luminometer is a laboratory instrument designed for the measurement of luminescence signals. It is capable of detecting and quantifying light emission from various luminescent assays, such as luciferase reporter gene assays and bioluminescence-based experiments.
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Lab products found in correlation
8 protocols using glomax dual luminometer
1
Cloning and Characterization of SOST Promoter
2
Estrogen and NF-κB Transcriptional Assay
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Cells were seeded in 24-well plates in replicates of six. Twenty-four hours after seeding, cells were transfected with 100 ng/well of a pGL3 luciferase reporter construct containing either estrogen response elements (ERE) or NFκB response elements (NREs) using FuGENE 6 (Promega, Madison, WI). One day after transfection, cells were treated with ethanol vehicle, 1 nM E2, 20 ng/ml TNFα, or E2 + TNFα for 24 h. Cells were washed once with PBS and subsequently lysed using 1X Passive Lysis Buffer (Promega). Equal amounts of protein lysate were assayed for luciferase activity after addition of Luciferase Assay Reagent using a Glomax-Dual Luminometer (Promega).
3
Estrogen receptor-mediated transcription assay
4
Luciferase Assay of Osteoblast Transfections
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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).
5
NFκB Transcriptional Activity Assay
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Cells were seeded in 24-well plates and treated with dox for 24 h prior to transfecting with 100 ng of a pGL3 luciferase reporter construct containing NFκB response elements (NRE) using FuGENE 6 (Roche, Basel, Switzerland). Twenty-four hours post-transfection, cells were treated with vehicle control, 1 nM E2, 20 ng/mL TNFα, or E2 + TNFα for an additional 24 h. Cells were washed once with 1X PBS and lysed using 1X Passive Lysis Buffer (Promega). Equal amounts of protein lysate were assessed for luciferase activity using Luciferase Assay Reagent and a Glomax-Dual Luminometer (Promega). Treatments were conducted in replicates of 6.
6
Osterix Promoter-Reporter Assay
7
Estrogen Receptor Beta Transactivation Assay
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MDA-MB-231-ERβ cells were plated at 35,000 cells/well in 24-well plates with 10% CS-FBS containing media supplemented with 100 ng/ml dox for 24 hours. Cells were transfected with 100 ng of the estrogen response element (ERE) luciferase reporter construct using FuGENE 6 transfection reagent (Roche) in CS-FBS containing media. The next day, cells were washed twice with 1X PBS and treated with CS-FBS containing media supplemented with 100 ng/ml Dox and ethanol control, 1 nM estrogen, 1, 10, 100, and 1000 nM LY500307 for 24 hours. Cells were harvested using 1X Passive Lysis Buffer (Promega, Madison, WI) and equal amounts of protein extract were assayed using luciferase assay reagent and a Glomax-Dual Luminometer (Promega).
8
Valve Interstitial Cells Luciferase Assay
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