Pgl4.74 renilla luciferase

Manufactured by Promega

PGL4.74 Renilla luciferase is a reporter gene that expresses the Renilla luciferase enzyme. Renilla luciferase is a naturally occurring enzyme that catalyzes the oxidation of coelenterazine, resulting in the emission of light.

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Lab products found in correlation

Flag keap1, by Addgene (1 mentions) Pcdna3 egfp c4 nrf2, by Addgene (1 mentions) Pcdna3.1 flag nrf2, by Addgene (1 mentions) Flag nrf2, by Addgene (1 mentions) Glomax detection system, by Promega (1 mentions) Dual glow luciferase assay kit, by Promega (1 mentions) Wallac victor, by PerkinElmer (1 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions) Dual luciferase reporter assay system, by Promega (1 mentions)

Close spelling variants of this product

PGL4.74 Renilla luciferase expression vector PGL4.74 TK-Renilla-luciferase Renilla luciferase pGL4.74 [hRluc/TK] plasmid PGL4.74 Renilla luciferase normalization control PGL4.74 Renilla Luciferase control vector PGL4.74 encoding Renilla luciferase Renilla luciferase

4 protocols using pgl4.74 renilla luciferase

Luciferase Assay for Wnt and SOX Signaling

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For luciferase assays, 1 × 10⁵ CC14 cells, naive or infected with the appropriate shRNAs, were plated in 24-well plates and then transfected with Lipofectamine LTX with a plasmid containing TOP/FOP reporters (500 ng), pGL4.74 Renilla luciferase (100 ng, Promega), pRFP as transfection control (100 ng), CMV-β-CATENIN or CMV-SOX4/SOX12/SOX17 as required. A pCMV-control was used to complement or replace the other expression plasmids in order to keep the amount of DNA and of the CMV promoter constant.

Duquet A., Melotti A., Mishra S., Malerba M., Seth C., Conod A, & Ruiz i Altaba A. (2014). A novel genome-wide in vivo screen for metastatic suppressors in human colon cancer identifies the positive WNT-TCF pathway modulators TMED3 and SOX12. EMBO Molecular Medicine, 6(7), 882-901.

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Plasmids for NRF2 and HIF1α Regulation

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For transient transfections the following plasmids were used: pGL3-8xARE^{42 (link)}, or pGLHIF1.3^{43 (link)}, pGL4.74 renilla luciferase (#E6921, Promega), pGFP-NRF2 (pcDNA3-EGFP-C4-Nrf2, #21549, Addgene), Flag-NRF2 (pCDNA3.1 FLAG NRF2, #36971, Addgene), Flag-Keap1 (#28023, Addgene), p(HA)HIF1alpha (P402A,P564A) (#52636, Addgene). To generate plasmid GFP-NRF2-S40A, a modified QuickChange protocol was used^{44 (link)} to perform a PCR using pGFP-NRF2 as the template and primers NRF2-S40A-for (5ʹ-GTCGAGAAGTATTTGACTTCGCCCAGCGACGGAAA-GAGTATGAGCTGGAAAA-3ʹ) and NRF2-S40A-rev (5ʹ-GGCGAAGTCAAATAC-TTCTCGACTTACTCCAAGA TCTATATCTTGCCTCCAAAGTATGTCA-3ʹ). pLKO.1-shRNA-NRF2 tet-on (tetracycline inducible) contained the sequence 5ʹ-GCTCCTACTGTGATGTGAAAT-3ʹ of NRF2 mRNA (GenBank acc. no. NM_001145412), pLKO.1-shRNA-PERK tet-on (tetracycline inducible) contained the sequence 5ʹ-GGAACGACCTGAAGCTATAAA-3ʹ of PERK mRNA (GenBank acc. no. NM_004836).

Küper A., Baumann J., Göpelt K., Baumann M., Sänger C., Metzen E., Kranz P, & Brockmeier U. (2021). Overcoming hypoxia-induced resistance of pancreatic and lung tumor cells by disrupting the PERK-NRF2-HIF-axis. Cell Death & Disease, 12(1), 82.

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Dual-Luciferase Transcriptional Assay

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5 × 10⁴ cells were seeded in 24-well plates. After 24 h, cells were transfected with 500 ng pGLHIF1.3^{47 (link)} and 100 ng pGL4.74 renilla luciferase (#E6921, Promega) per well. After incubation for further 72 h, cells were lysed and prepared using the Dual Glow luciferase assay kit (Promega). Firefly and renilla luciferase were measured using the GloMax detection system (Promega) and normalized to renilla values to exclude variations in transfection.

Ocklenburg T., Neumann F., Wolf A., Vogel J., Göpelt K., Baumann M., Baumann J., Kranz P., Metzen E, & Brockmeier U. (2021). In oxygen-deprived tumor cells ERp57 provides radioprotection and ensures proliferation via c-Myc, PLK1 and the AKT pathway. Scientific Reports, 11, 7199.

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Transient Transfection Assay for NR5A1 Function

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Transient gene expression assays for the assessment of NR5A1 function were performed in 96-well plates with the use of HEK 293T cells transfected at 70% confluence, Lipofectamine 2000 reagent (Invitrogen), and a dual-Luciferase reporter assay system (Promega) with pGL4.74 Renilla luciferase (Promega) expression as a marker of transfection efficiency. Then pCMV6_WT or mutant NR5A1 expression vectors (10 ng per well) were cotransfected into HEK 293T cells with NR5A1-responsive minimal promoters linked to luciferase (human CYP11A1, human CYP17A1; 10 ng per well). Cells were lysed 48 hours later, and luciferase assays were performed with the use of a multilabel plate reader (Wallac Victor, Perkin-Elmer). All data were standardized for Renilla activity. Results are shown as the mean AE SEM of three independent experiments, each performed in triplicate. The data were analyzed statistically using Student's t test. A P value of < .05 was considered statistically significant.

Rocca M.S., Ortolano R., Menabò S., Baronio F., Cassio A., Russo G., Balsamo A., Ferlin A., Ferlin A, & Baldazzi L. (2018). Mutational and functional studies on NR5A1 gene in 46,XY disorders of sex development: identification of six novel loss of function mutations. Fertility and sterility, 109(6).

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