plasmid mixture  (Promega)

Journal: PLoS ONE

Article Title: ABCG1 rs57137919G>A Polymorphism Is Functionally Associated with Varying Gene Expression and Apoptosis of Macrophages

doi: 10.1371/journal.pone.0097044

Figure Lengend Snippet: (a) Schematic diagram for constructing wild-type or mutant promoter at the ABCG1 rs57137919G>A polymorphism site. Two constructs were subcloned into pGL3-basic plasmid vectors with firefly luciferase reporter gene. pG, -367G (open bar); pA, -367A (grey bar). Luciferase activity assays for two ABCG1 promoter constructs in HEK293T cells (b), THP-1 cells (c), and HepG2 cells (d) are shown. Luciferase activities were measured with the dual-luciferase reporter assay system and normalized to Renilla luciferase activity 24 hours after transfection in the presence or absence of TO901317. The luciferase activity was a corrected relative value. Data are shown as mean ± SD of four independent experiments in triplicate. P <0.01 vs. pG in HEK293 cells, THP-1 cells, and HepG2 cells under both basal state and LXR agonist stimulation.

Article Snippet: The pGL3-basic empty vector was used as a negative control and the pGL3-control vector (Promega) was used as a positive control for the luciferase assay.

Techniques: Mutagenesis, Construct, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay, Transfection

Journal: Oncology Letters

Article Title: Significant association of EED promoter hypomethylation with colorectal cancer

doi: 10.3892/ol.2019.10432

Figure Lengend Snippet: Dual-luciferase reporter assay in the 293T cell line. The pGL3-Basic and pGL3-Promoter vectors were used as negative and positive controls, respectively. The relative luciferase level is displayed for pGL3-Promoter, pGL3-Basic and pGL3-EED The pGL3-EED indicates the recombinant EED fragment ligated to the pGL3-Basic vector. An analysis of variance and Bonferroni's correction were applied for statistical analysis. P<0.05 was considered to indicate a statistically significant difference. *P<0.0001. EED, embryonic ectoderm development; Luc, luciferase.

Article Snippet: The empty pGL3 basic vector was used as the negative control and the pGL3 promoter vector (both Promega Cooperation, Madison, WI, USA) was used as the positive control, which contained an SV40 promoter upstream of the luciferase gene.

Techniques: Luciferase, Reporter Assay, Recombinant, Plasmid Preparation

Journal: Scientific Reports

Article Title: Elevated UMOD methylation level in peripheral blood is associated with gout risk

doi: 10.1038/s41598-017-11627-w

Figure Lengend Snippet: Dual-luciferase reporter assay in HEK-293T cell line. The pGL3 Basic and promoter vectors were used as negative and positive control in this study, respectively. Relative luciferase activity was performed in triplicates.

Article Snippet: The empty pGL3-Basic vector was used as negative control, and the pGL3-Control vector, (Promega, Madison city, WI, USA) containing an SV40 promoter upstream of the luciferase gene was used as positive control.

Techniques: Luciferase, Reporter Assay, Positive Control, Activity Assay

Journal: Purinergic Signalling

Article Title: Insulin requires A 1 adenosine receptors expression to reverse gestational diabetes-increased L-arginine transport in human umbilical vein endothelium

doi: 10.1007/s11302-015-9491-2

Figure Lengend Snippet: Involvement of A1AR and A2AAR on GDM and insulin effect on hCAT-1 expression. a Western blot for hCAT-1 protein abundance in HUVECs from normal (Normal) or gestational diabetes mellitus (GDM) pregnancies incubated in the absence (−) or presence (+) insulin (1 nM, 8 h) and/or A1 adenosine receptor (A1AR) antagonist (Antag). Lower panel, hCAT-1/β-actin ratio densitometries normalized to 1 in cells from normal pregnancies in the absence of insulin or the antagonist. b hCAT-1 protein abundance as in a for A2AAR antagonist. c hCAT-1 mRNA expression as in a for A1AR antagonist. d hCAT-1 mRNA expression as in b for A2AAR antagonist. e Luciferase (Luc) reporter constructs containing two truncations of SLC7A1 promoter (−1606 and −650 bp from the transcription start point) were transfected in HUVECs from normal or GDM pregnancies, along with Renilla reporter plasmid, and assayed for Firefly and Renilla luciferase activity, respectively. Results depict ratio of Firefly/Renilla luciferase activity. After 36 h of transfection, cells were incubated for further 8 h without (Without insulin) or with (With insulin) insulin (1 nM) in the absence (Control) or presence of A1AR or A2AAR antagonists. Cells were also transfected with the empty pGL3-basic vector or pGL3-control vector (SV40 pGL3) as negative or positive controls, respectively (see “Materials and methods”). In a and c, *P < 0.05 vs. corresponding values in Normal. †P < 0.05 vs. values in the absence of insulin or A1AR antagonist. ‡P < 0.05 vs. all other corresponding values. In b and d, *P < 0.05 vs. corresponding values in Normal. †P < 0.05 vs. all other corresponding values. In e, *P < 0.05 vs. vs. all other values except between themselves in the corresponding promoter constructs. Values are mean ± SEM (n = 38)

Article Snippet: Aliquots of cell suspension (0.5 mL, 3.2 × 10 6 cells/mL) were mixed with 10 μg of pGL3–hCAT1 −1606 or pGL3–hCAT1 −650 constructs, pGL3-Basic (empty pGL3 vector), pGL3-Control (Simian Virus 40 promoter (SV40) pGL3 vector), and the internal transfection control vector pRL-TK expressing Renilla luciferase (Promega) [ 6 ].

Techniques: Expressing, Western Blot, Incubation, Luciferase, Construct, Transfection, Plasmid Preparation, Activity Assay

Journal: Purinergic Signalling

Article Title: Insulin requires A 1 adenosine receptors expression to reverse gestational diabetes-increased L-arginine transport in human umbilical vein endothelium

doi: 10.1007/s11302-015-9491-2

Figure Lengend Snippet: GDM and insulin effect on hCAT-1 expression in A1AR and A2AAR knockdown cells. a Western blot for hCAT-1 protein abundance in HUVECs in the absence (−) or presence (+) of insulin (1 nM, 8 h) in non-transfected (−) or transfected (+) cells with siRNA against A1AR (KDA1AR). Lower panel, hCAT-1/β-actin ratio densitometries normalized to 1 in cells transfected with sc-siRNA from normal or GDM pregnancies in the absence of insulin. b Western blot for hCAT-1 protein abundance with siRNA against A2AAR (KDA2AAR) as in A. c hCAT-1 mRNA expression in KDA1AR or KDA2AAR cells as in A. d Luciferase (Luc) reporter construct pGL3-hCAT-1−1606 of SLC7A1 promoter transfected in KDA1AR or KDA2AAR cells, along with Renilla reporter plasmid. After 36 h of transfection, cells were incubated without (−) or with (+) insulin (1 nM, 8 h) (see “Materials and methods”). e Luciferase (Luc) reporter construct SLC7A1 promoter transfected in KDA1AR or KDA2AAR cells as in d. In a, *P < 0.05 vs. all other values except between themselves, †P < 0.05 vs. all other corresponding values in GDM. In b, *P < 0.05 vs. all other values. †P < 0.05 vs. all other values except between themselves. In c–e, *P < 0.05 vs. all other values in Normal except between themselves. †P < 0.05 vs. all other values in GDM except between themselves. ‡P < 0.05 vs. corresponding values in Normal. Values are mean ± SEM (n = 29)

Article Snippet: Aliquots of cell suspension (0.5 mL, 3.2 × 10 6 cells/mL) were mixed with 10 μg of pGL3–hCAT1 −1606 or pGL3–hCAT1 −650 constructs, pGL3-Basic (empty pGL3 vector), pGL3-Control (Simian Virus 40 promoter (SV40) pGL3 vector), and the internal transfection control vector pRL-TK expressing Renilla luciferase (Promega) [ 6 ].

Techniques: Expressing, Western Blot, Transfection, Luciferase, Construct, Plasmid Preparation, Incubation