The firefly luciferase reporter for Sel1l or Hrd1 (Syvn1) promoter was constructed by cloning the genomic region into the MluI and XhoI sites or the MluI and HindIII sites in the pGL-3 basic vector (Promega), respectively. Mutations were made by overlap extension polymerase chain reaction as previously described (Bryksin and Matsumura, 2010 (link)). All constructs were verified by DNA sequencing. The sequences of all primers are listed in Appendix 1. HEK293T cells were transfected with Sel1l or Hrd1 promoter constructs, pRL-PGK (Promega) and 3xFlag-NICD1 (Addgene, #20183) or empty using Lipofectamine 3000 (Invitrogen, L3000015). Cell lysates were collected 48 hr after transfection, and luciferase activities were analyzed using the dual-luciferase reporter assay system (E1910, Promega). pRL-PGK, which expresses Renilla luciferase, was used as the internal control for adjustment of discrepancies in transfection and harvest efficiencies. EL4 cells were transfected with Sel1l or Hrd1 promoter constructs and pRL-PGK (Promega) using Lipofectamine 3000 (Invitrogen, L3000015). Cells were incubated with PBS or 5 μg/ml recombinant mouse DLL4 (BioLegend, #776706) for 24 hr before analysis.
Prl pgk
Manufactured by Promega
The PRL-PGK is a lab instrument that measures the activity of the enzyme phosphoglycerate kinase (PGK). It is used to analyze the metabolic activity of cells or biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (4 mentions)
Recombinant mouse dll4, by BioLegend (2 mentions)
3xflagnicd1, by Addgene (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Pgl3 basic vector, by Promega (2 mentions)
Psicheck2 dual luciferase vector, by Promega (1 mentions)
Pnf κb luc, by Takara Bio (1 mentions)
4 protocols using prl pgk
1
Luciferase Reporter Assay for Sel1l and Hrd1 Promoters
2
Luciferase Reporter for Sel1l and Hrd1 Promoters
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The firefly luciferase reporter for Sel1l or Hrd1 (Syvn1) promoter was constructed by cloning the genomic region into the MluI and XhoI sites or the MluI and HindIII sites in the pGL-3 basic vector (Promega), respectively. Mutations were made by overlap extension polymerase chain reaction as previously described (Bryksin and Matsumura, 2010) (link). All constructs were verified by DNA sequencing. The sequences of all primers are listed in Supplemental Table 2. 293T cells were transfected with Sel1l or Hrd1 promoter constructs, pRL-PGK (Promega) and 3xFlag-NICD1 (Addgene, #20183) or empty using Lipofectamine 3000 (Invitrogen, L3000015). Cell lysates were collected 48 hours after transfection, and luciferase activities were analyzed using the dual-luciferase reporter assay system (E1910, Promega). pRL-PGK, which expresses Renilla luciferase, was used as the internal control for adjustment of discrepancies in transfection and harvest efficiencies. EL4 cells were transfected with Sel1l or Hrd1 promoter constructs and pRL-PGK (Promega) using Lipofectamine 3000 (Invitrogen, L3000015). Cells were incubated with PBS or 5 µg/ml recombinant mouse DLL4 (BioLegend, #776706) for 24 hours before analysis.
3
Validating miR-195-3p Target Genes via Luciferase Assay
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The wild-type or mutant fragment of TINCR or ST6GAL1 containing the predicting binding sequence of miR-195-3p was subcloned into a psiCHECK2 Dual-luciferase vector (Promega). To verify the target genes of miRNAs, HCC cells were co-transfected with luciferase reporter plasmids and miR-195-3p mimics or negative control. Luciferase activity was measured using the dual-luciferase reporter assay system (Promega). Renilla luciferase expressed by pRL-PGK (Promega) was used as an internal control to correct for differences in both transfection and harvest efficiency.
4
Luciferase-Based Assays for Studying NF-κB, miRNA, and ceRNA Mechanisms
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Luciferase activity was measured using the dual‐luciferase reporter assay system (Promega). Renilla luciferase expressed by pRL‐PGK (Promega) was used as an internal control to correct for differences in both transfection and harvest efficiency.
To examine the activity of NF‐κB signaling, a luciferase reporter plasmid containing the minimal promoter with multiple tandem NF‐κB‐binding sites (pNF‐κB‐Luc; Clontech) was used. Cells were transfected with 50 nM RNA duplex for 24 hours and then co‐transfected with 50 ng pNF‐κB‐Luc and 2 ng pRL‐PGK for 32 hours, followed by Dox treatment for 12 hours before the luciferase activity assay.
To verify the target genes of miRNAs, cells were co‐transfected with 50 nM miRNAs or NC duplex, 2 ng pRL‐PGK, and 20 ng firefly luciferase reporter plasmid that contained the wild‐type or mutant miRNA‐binding sequence of the target gene for 48 hours.
To test the competing endogenous RNA (ceRNA) activity of PDIA3P1 with the luciferase reporter system, SK‐PDIA3P1, SK‐Ctrl, QGY‐PDIA3P1, or QGY‐Ctrl cells were co‐transfected with 10 nM RNA duplex, 2 ng pRL‐PGK, and 10 ng pGL3cm‐TRAF6‐3′ UTR.
To characterize the PDIA3P1 promoter, cells were co‐transfected with 2 ng pRL‐PGK and 100 ng pGL3‐basic‐p‐(−2,129/+358) for 48 hours.
To examine the activity of NF‐κB signaling, a luciferase reporter plasmid containing the minimal promoter with multiple tandem NF‐κB‐binding sites (pNF‐κB‐Luc; Clontech) was used. Cells were transfected with 50 nM RNA duplex for 24 hours and then co‐transfected with 50 ng pNF‐κB‐Luc and 2 ng pRL‐PGK for 32 hours, followed by Dox treatment for 12 hours before the luciferase activity assay.
To verify the target genes of miRNAs, cells were co‐transfected with 50 nM miRNAs or NC duplex, 2 ng pRL‐PGK, and 20 ng firefly luciferase reporter plasmid that contained the wild‐type or mutant miRNA‐binding sequence of the target gene for 48 hours.
To test the competing endogenous RNA (ceRNA) activity of PDIA3P1 with the luciferase reporter system, SK‐PDIA3P1, SK‐Ctrl, QGY‐PDIA3P1, or QGY‐Ctrl cells were co‐transfected with 10 nM RNA duplex, 2 ng pRL‐PGK, and 10 ng pGL3cm‐TRAF6‐3′ UTR.
To characterize the PDIA3P1 promoter, cells were co‐transfected with 2 ng pRL‐PGK and 100 ng pGL3‐basic‐p‐(−2,129/+358) for 48 hours.
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