CHO and HepG2 cells were transfected with the indicated pBIND vector together with the pG5luc reporter plasmid (Promega) which contains five GAL4 binding sites upstream of a minimal TATA box using TransIT LT-1 transfection reagent (Takara). At 24 hours after transfection, the cells were harvested and plated in a 96-well tissue culture plate. The cells were subsequently treated with each compound and, after 24 hours of treatment, were lysed in lysis buffer (Promega) and analyzed using the Dual-Luciferase® Reporter Assay System (Promega). The Firefly luciferase signal was normalized to the Renilla luciferase signal.
Pg5luc reporter plasmid
Manufactured by Promega
The PG5luc reporter plasmid is a DNA construct used in gene expression studies. It contains a luciferase reporter gene that can be used to measure the activity of a promoter of interest. The plasmid can be transfected into cells, and the luciferase activity can be quantified to assess the expression of the target gene.
Automatically generated - may contain errors
2 protocols using pg5luc reporter plasmid
1
Dual-Luciferase Assay for Transcriptional Activity
2
Plasmid Construction for Transcription Factor Studies
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The HA-tagged GCM1 expression plasmid, pHA-GCM1, has been described previously19 (link). A DNA fragment encoding human GATA3 with an N-terminal triple HA tag or a C-terminal triple FLAG tag was subcloned into a CMV or EF1 promoter-driven expression vector to generate pHA-GATA3 or pGATA3-FLAG. The HtrA4 promoter region from nt −971 to 29 (relative to the translational initiation site) was subcloned into pGL3E1B to generate the pHtrA4-1 kb reporter plasmid. The p(GBS)4E1bLuc reporter plasmid was generated by subcloning four copies of the proximal GCM1-binding site derived from the syncytin-1 gene in the pGL3E1B luciferase reporter vector. The pG5-Luc reporter plasmid which harbors five copies of the Gal4-binding site was obtained from Promega (Madison, WI).
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