HepG2 cells were seeded in 24-well plates with 10% sFBS medium overnight. A total of 0.5 μg of DNA, including 0.2 μg of WT ERα or Q375 mutants, 0.2 μg of 3xERE Luc, and 0.1 μg of pRL-TK Luc plasmids, was transfected into the cells using Effectene transfection reagent (Qiagen). After 6 hours, cells were changed to fresh 10% sFBS medium overnight and then treated with vehicle control (DMSO, final concentration < 0.01%), E2, CME, Et-E2, DES, Hex, or THC (0.01 to 100 nM) for 18 hours. Luciferase assays were performed using the Dual Luciferase Reporter Activity System (Promega). Transfection efficiency was normalized against the renilla luciferase. Fold changes were calculated relative to vehicle controls. All experiments were repeated at least three times. Data shown are the average of triplicate determinations in a representative experiment. Values were calculated relative to vehicle control and presented as ±SEM.
Dual luciferase activity reporter system
Manufactured by Promega
Sourced in United States
The Dual-Luciferase Activity Reporter System is a laboratory instrument that simultaneously measures the activities of two different luciferase reporter enzymes within a single sample. It provides a quantitative analysis of gene expression and cellular events.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
White flat bottom 96 well plate, by Corning (1 mentions)
Neon transfection system 10 l kit, by Thermo Fisher Scientific (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Z ietd fmk, by R&D Systems (1 mentions)
Panc 1, by Thermo Fisher Scientific (1 mentions)
Anti dr5, by ProSci (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Suit 2, by Thermo Fisher Scientific (1 mentions)
Trifluoperazine tfp, by Merck Group (1 mentions)
Anti caspase 8, by BD (1 mentions)
Tamoxifen tmx, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Protein g agarose, by Thermo Fisher Scientific (1 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
Suit 2, by American Type Culture Collection (1 mentions)
Pmirglo, by Promega (1 mentions)
Pmirglo dual luciferase vector, by Promega (1 mentions)
Site direct mutagenesis kit, by Takara Bio (1 mentions)
8 protocols using dual luciferase activity reporter system
1
ER Transactivation Assay in HepG2 Cells
2
Notch Pathway Transcriptional Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Notch pathway function was analyzed by measuring the transcriptional activity of its downstream component RBP-jk using a Cignal RBP-Jk Dual Luciferase Reporter assay (Qiagen, Germantown, MD; # 336841). MCF-10A cells were transfected with Transcription Factor Reporter, Negative Control reporter or Positive Control constructs using the Neon Transfection System 10 µl kit (Invitrogen, Walthan, MA; # MPK1025). Twenty-four hours after transfection, cells were exposed to Sodium octanoate (OA, 5 mM) dissolved in PBS or PBS for 24 h. Cells were then lysed using Passive Lysis Buffer (Promega, Madison, WI) and transferred to a 96-well white flat-bottom plate (Corning, Tewksbury, MA). Luciferase activity was measured with the Dual-Luciferase Reporter Activity system (Promega; # E1910) using a Biotek Cytation 3 multiwell reader. Firefly luciferase activity was normalized to Renilla luciferase activity. All transfections were performed in triplicate. Luciferase measurement for each biological replicate was performed in three technical replicates. Values are expressed as mean ± SEM. The p-value was calculated by unpaired t-test.
3
miR-584-5p Binds KCNE2 3'-UTR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Targets of miR‐584‐5p was predicted by TargetScan. Among all these predicted targets, KCNE2 was selected for further investigation. The wild‐type or mutant 3′‐UTR of KCNE2 was cloned into a luciferase activity named pGL3 (Promega, Madison, WI). These vectors were designated as wt‐KCNE2 or mt‐KCNE2, respectively. Cells were then c‐transfected with wt‐KCNE2 or mt‐KCNE2 and miR‐584‐5p inhibitor or miR‐NC using Lipofectamine 2000. Relative luciferase activity was measured with dual‐luciferase activity reporter system (Promega) after transfection for 48 hr.
4
Pancreatic Cancer Cell Lines: Cultivation and Drug Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human pancreatic cancer cell lines PANC-1 and Suit-2 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). PANC-1 cells were grown in Dulbeccos' Modified Eagle's Media (DMEM; Invitrogen, Carlsbad, CA)) and Suit 2 cells were grown in RPMI 1640 supplemented with penicillin (5 units/mL), streptomycin (5 μg/mL), and 10% heat-inactivated FBS.
DR5 agonist antibody, TRA-8, was generated as previously described [48 ]. All antibodies used were commercially available, including anti-caspase-8 (BD Bioscience, San Jose, CA), anti-caspase-3 (Enzo Life, Plymouth Meeting, PA), anti-FADD and anti-CaM (Millipore, Billerica, MA), Src (Cell Signaling Technology, Danvers, MA), anti-DR5 (Prosci, Poway, CA), anti-c-FLIP (Enzo Life, Farmingdale, NY) and anti-GAPDH (Santa Cruz Biotech, Santa Cruz, CA).
Tamoxifen (TMX) and Trifluoperazine (TFP) were obtained from Sigma-Aldrich (St. Louis, MO). Protein G-agarose and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA). Caspase-8 Inhibitor, Z-IETD-FMK, was from R&D system. The dual-luciferase activity reporter system was purchased from Promega.
DR5 agonist antibody, TRA-8, was generated as previously described [48 ]. All antibodies used were commercially available, including anti-caspase-8 (BD Bioscience, San Jose, CA), anti-caspase-3 (Enzo Life, Plymouth Meeting, PA), anti-FADD and anti-CaM (Millipore, Billerica, MA), Src (Cell Signaling Technology, Danvers, MA), anti-DR5 (Prosci, Poway, CA), anti-c-FLIP (Enzo Life, Farmingdale, NY) and anti-GAPDH (Santa Cruz Biotech, Santa Cruz, CA).
Tamoxifen (TMX) and Trifluoperazine (TFP) were obtained from Sigma-Aldrich (St. Louis, MO). Protein G-agarose and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA). Caspase-8 Inhibitor, Z-IETD-FMK, was from R&D system. The dual-luciferase activity reporter system was purchased from Promega.
5
CEACAM1 3'UTR Binding Sites Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sequences of 3’UTR of CEACAM1 contained binding sites (CACUGCC) with miR-449a were amplified and inserted into luciferase reporter plasmids to construct the wild-type recombinational luciferase reporter genes (CEACAM1-WT). In addition, the binding site sequences were mutated into CUGACGC, and the mutant-type recombinational luciferase reporter genes (CEACAM1-MUT) were constructed. Then, the recombinational wild/mutant type luciferase reporters were con-transfected with miR-499a mimic or its negative control into HUVECs and incubated for 48 h. After that, the luciferase activity was measured using a dual-luciferase activity reporter system (Promega, Madison, WI, USA) according to the manufacturer’s protocol.
6
Predicting miRNA-mRNA Interactions in Rectal Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ENCORI website was used to predict the miRNA targets of SNHG17 and also the messenger RNA target of miRNA. After prediction, miR-519c-3p and STC2 were selected for the following analyses with the following criteria: (1) the miRNA expression level was downregulated, while miRNA target gene expression level was upregulated in tumor tissues; and (2) that targets have roles in carcinogenesis based on previous studies. The 3'-UTR of wild-type sequences of SNHG17 or STC2 (wt/mt-SNHG17/STC2) were inserted into pmiR-GLO (Promega, Beijing, China). Then, the vectors were transfected into rectal cancer cells along with miRNAs using Lipofectamine 2000. After transfection for 48 h, relative luciferase activity was measured with the Dual-luciferase activity reporter system (Promega).
7
LncRNA-miRNA Interaction Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DIANA LncBase Predicted V.2 (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2%2Findex-predicted
8
Validating miR-98-5p Binding to IGF1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 3'-UTR contains the binding site for hsa-miR-98-5p in IGF1 was ampli ed from genome and cloned into pGL3 vector (Promega, Madison, WI, USA) and named as wt-IGF1. The mutant IGF1 luciferase vector (mt-IGF1) was constructed from wt-IGF1 using site-direct mutagenesis kit (Takara). Cells were cotransfected with wt-IGF1 or mt-IGF1 and hsa-miR-98-5p mimic or negative control miRNA (miR-NC) using Lipofectamine 2000 (Invitrogen). The hsa-miR-98-5p mimic or miR-NC were purchased from RiboBio (Guangzhou, China). After co-transfection for 48 h, relative luciferase activity was measured using dualluciferase activity reporter system (Promega).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!