pgl3-enhancer firefly luciferase reporter vector  (Promega)

Journal: Journal of Molecular Endocrinology

Article Title: CEBPB/POU2F2 modulates endothelin 1 expression in prehypertensive SHR vascular smooth muscle cells

doi: 10.1530/JME-22-0178

Figure Lengend Snippet: Analysis of the enhancer region of the rat endothelin promoter in vascular smooth muscle cells (VSMCs). (A) The region −1309/+0 and deletion at 5' DNA of rat endothelin ( ET ) -1 were cloned in the pGL3-enhancer Firefly luciferase reporter vector and co-transfected with a pRL-CMV-derived Renilla luciferase reporter plasmid in spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) VSMCs. Transcriptional activity was normalized to the level of Renilla activity and expressed as fold activity of 1309prET1 in WKY rats. Smooth muscle cells were collected from the aortas of three SHR or WKY rats, and each data set consists of three experiments. (B) Sequence alignment between −1309 to −1079 upstream of the Edn1 promoter in Sprague–Dawley (SD) rats, SHRs, and WKY rats. The underlined sequence region is the putative binding site for POU2F2 and CEBPB. (C) Sequence logo of two major putative elements on the Edn1 promoter enhancer region. These logos were compiled by JASPAR software from published human cellular transcription factor-binding sites. Statistical data are shown as the mean ± s.e.m. , * P < 0.05, ## P < 0.01. Student's t -test from three independent experiments. *vs 1309prET1 in WKY, # vs 81prET1 in WKY rats. A full color version of this figure is available at https://doi.org/10.1530/JME-22-0178 .

Article Snippet: The 1309prET1 plasmid was inserted into pGL3-enhancer with firefly luciferase reporter gene vector (Promega) at the XhoI-HindIII cloning site.

Techniques: Clone Assay, Luciferase, Plasmid Preparation, Transfection, Derivative Assay, Activity Assay, Sequencing, Binding Assay, Software

Journal: Journal of Cancer

Article Title: Cloning and characterization of the putative AFAP1-AS1 promoter region

doi: 10.7150/jca.29049

Figure Lengend Snippet: Primer pairs used for generating AFAP1-AS1 promoter deletion constructs.

Article Snippet: All of the primers included 15-bp sequences homologous to the pGL3-enhancer luciferase reporter vector (Promega, Madison, WI, USA).

Techniques: Construct

Journal: Journal of Cancer

Article Title: Cloning and characterization of the putative AFAP1-AS1 promoter region

doi: 10.7150/jca.29049

Figure Lengend Snippet: Construction of the reporter vector for the -1521/+220 region of the AFAP1-AS1 promoter. A, Gel analysis of the amplification of the -1521/+220 region, lane 1: DL2000 DNA marker, lane 2: the amplification of the -1521/+220 region detected by 1% agarose gel. B, Sequencing results of the pGL3-1521/+220 reporter construct. C, The pGL3-1521/+220 reporter plasmid and pRL-TK were transiently co-transfected into HNE2 cells. Luciferase activities were measured 48 h after transfection. The results are presented as the relative luciferase activity. Positive control: pGL3-control vector, negative control: pGL3-enhancer vector. The deletion samples were compared to the negative control. Data are shown as the means ± SD of at least three independent experiments, *** P < 0.001.

Article Snippet: All of the primers included 15-bp sequences homologous to the pGL3-enhancer luciferase reporter vector (Promega, Madison, WI, USA).

Techniques: Plasmid Preparation, Amplification, Marker, Agarose Gel Electrophoresis, Sequencing, Construct, Transfection, Luciferase, Activity Assay, Positive Control, Negative Control

Journal: Journal of Cancer

Article Title: Cloning and characterization of the putative AFAP1-AS1 promoter region

doi: 10.7150/jca.29049

Figure Lengend Snippet: Luciferase reporter assay for the AFAP1-AS1 promoter. A, Schematic illustration of the 5'-deletion and 3'-deletion constructs of the AFAP1-AS1 promoter. B, The pGL3-enhancer and the deletion constructs were transiently transfected into HNE2 cells with pRL-TK. Luciferase activities were measured 48 h after transfection. The results are presented as the relative luciferase activity. Positive control: pGL3-control vector, negative control: pGL3-enhancer vector. The deletion samples were compared to the negative control. Data are shown as the means ± SD of at least three independent experiments. *** P < 0.001, ns: no significance.

Article Snippet: All of the primers included 15-bp sequences homologous to the pGL3-enhancer luciferase reporter vector (Promega, Madison, WI, USA).

Techniques: Luciferase, Reporter Assay, Construct, Transfection, Activity Assay, Positive Control, Plasmid Preparation, Negative Control