Hre luc

Manufactured by Addgene

The HRE-Luc is a reporter construct that contains hypoxia response elements (HREs) coupled to a luciferase reporter gene. It can be used to monitor and quantify the activation of hypoxia-inducible factor (HIF) signaling in cells.

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Lab products found in correlation

Ha hif1α, by Addgene (2 mentions) Dual luciferase reporter assay system, by Promega (2 mentions) Ha hif 1α 18949, by Addgene (2 mentions) Ha hif 1α, by Takara Bio (2 mentions) Lv shhif 1α, by Merck Group (1 mentions) Pspax2, by Addgene (1 mentions) Pmd2 g, by Addgene (1 mentions) Hdac4, by Addgene (1 mentions) Odd luciferase pcdna3, by Addgene (1 mentions) Prl tk, by Promega (1 mentions) Pcmv renilla, by Promega (1 mentions) Synergy 2 plate reader, by Agilent Technologies (1 mentions) X tremegene 9, by Roche (1 mentions) Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Pnf κb luc, by Addgene (1 mentions) Iκbα 44d4 4812, by Cell Signaling Technology (1 mentions) Cell culture products, by Thermo Fisher Scientific (1 mentions)

8 protocols using hre luc

Hypoxia-Inducible Factor Manipulation

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LV-shHIF-1α (#232222 designated clone 1, #54450 designated clone 2) and control pLKO.1 were purchased from Sigma-Aldrich. siHIF-1α was a gift from Dr. Connie Cepko (Addgene). The following plasmids were obtained from the Addgene repository: HRE-Luc (#26731) developed by Navdeep Chandel, HA-HIF1α (#18949) developed by Dr. William Kaelin, psPAX2 (catalogue no. 12260) and pMD2G (catalogue no. 12259) developed by Dr. Didier Trono.

Silagi E.S., Schoepflin Z.R., Seifert E.L., Merceron C., Schipani E., Shapiro I.M, & Risbud M.V. (2017). Bicarbonate Recycling by HIF-1-dependent Carbonic Anhydrase isoforms 9 and 12 is Critical in Maintaining Intracellular pH and Viability of Nucleus Pulposus Cells. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 33(2), 338-355.

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Transactivation Assays for HIF and p300

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For transactivation studies of HIF-1α, HIF-2α, and p300, the binary Gal4 reporter plasmids (HIF-1α-N-TAD, aa 530-778; HIF-1α-C-TAD, aa 740-826; HIF-2α-TAD, aa 819-870; p300-N-TAD, aa 1-596; p300-C-TAD, aa 1737-2414) were provided by Dr. Nianli Sang, Drexel University College of Medicine. pFR-Luc (Stratagene) reporter contains the yeast Gal4-binding site upstream of a minimal promoter and the Firefly luciferase gene. Enolase1-WT and Enolase1-HRE-mut promoter were provided by Dr. Gregg Semenza, Johns Hopkins University. HDAC1 expression construct was provided by Dr. Stuart Schreiber, Harvard University (22 (link)). HDAC2 and HDAC3 were provided by Dr. Ed Seto, H. Lee Moffitt Cancer Center Research Institute (23 (link), 24 (link)). HRE-Luc (#26731) by Navdeep Chandel; HDAC4 (#30485), HDAC6 (#30482) and HDAC6-DC (#30483) by Tso-Pang Yao, and ODD-luciferase-pcDNA3 by William Kaelin (#18956) were obtained from Addgene. pRLTK (Promega) containing the Renilla reniformis luciferase gene was used as an internal transfection control. PHD2^f/f;CreER(+) and PHD2^+/+;CreER(+) MEFs were a kind gift from Dr. William G. Kaelin of Harvard Medical School (25 (link)).

Schoepflin Z.R., Shapiro I.M, & Risbud M.V. (2016). Class I and IIa HDACs mediate HIF-1α stability through PHD2-dependent mechanism while HDAC6, a class IIb member, promotes HIF-1α transcriptional activity in nucleus pulposus cells of the intervertebral disc. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 31(6), 1287-1299.

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Hypoxia-Responsive Luciferase Assay

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DLK-S expression plasmid was kindly provided by prof. Anne Ferguson-Smith [11] (link), cloned into RCAS vector by classic restriction enzyme technique and transfected into DF-1 cells. For luciferase reporter assay, cells were co-transfected with hypoxia-responsive element (HRE)-luc (Addgene) [26] (link) or 8xCSL-luc (gift from Håkan Axelson) and pCMV-renilla (Promega) and analyzed using the Dual-Luciferase Reporter Assay System (Promega) on a Synergy 2 platereader (BioTek). Xtreme gene 9 (Roche) reagent was used according to manufacturer's recommendations for transient transfections.

Grassi E.S., Jeannot P., Pantazopoulou V., Berg T.J, & Pietras A. (2020). Niche-derived soluble DLK1 promotes glioma growth. Neoplasia (New York, N.Y.), 22(12), 689-701.

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Generating Mutant HIF-1α and PIM1 Plasmids

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HA-HIF-1α (18949) and HRE-Luc were purchased from Addgene. HA-HIF-1α-T455A and HA-HIF-1α-T455D mutant plasmids were generated using site directed mutagenesis (Clontech). HA-PIM1 and HA-PIM1-K67M were gifts from Dr. Andrew S Kraft (UA). hPMI1 (short) was cloned into pCIP (lentiviral backbone) for generation of the hPIM1 cell lines, and PIM1 in FUCRW (RFP-lentiviral backbone) for generation of RFP-PIM1 cell lines. HA-Ub was a gift from Dr. Alexandra Newton (UCSD).

Casillas A.L., Chauhan S.S., Toth R.K., Sainz A.G., Clements A.N., Jensen C.C., Langlais P.R., Miranti C.K., Cress A.E, & Warfel N.A. (2021). Direct phosphorylation and stabilization of HIF-1α by PIM1 kinase drives angiogenesis in solid tumors. Oncogene, 40(32), 5142-5152.

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Plasmid Constructs for HIF-1α Assays

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HA-HIF-1α (28 (link)), HRE-Luc (29 (link)), ODD-Luc (30 (link)), CMV-Luc, and SV40-Luc (31 (link)) constructs were purchased from Addgene. The ODD-Luc (P564A) mutant was created using a Quikchange site directed mutagenesis kit (Agilent). Renilla-Luc (pRL4-Luc) was purchased from Promega. EGFP-HIF-1 α and HA-Ubiquitin were gifts from Dr. Wafik El Deiry (Fox Chase Cancer Center) and Dr. Alexandra Newton (UCSD), respectively.

Casillas A.L., Toth R.K., Sainz A.G., Singh N., Desai A.A., Kraft A.S, & Warfel N.A. (2017). Hypoxia-inducible PIM kinase expression promotes resistance to anti-angiogenic agents. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(1), 169-180.

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Generating Mutant HIF-1α and PIM1 Plasmids

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Hypoxia-Induced HIF1α Activity Assay

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Primary chondrocytes were seeded in 24-well plates and transfected with 0.5 μg of HIF1α-responsive element luciferase reporter vector (HRE-luc, Addgene, #26731) [28 (link)] and 0.05 μg of a Renilla luciferase reporter vector using Lipofectamine 3000 (Invitrogen). After transfection for 24 h, chondrocytes were cultured under normoxia or hypoxia for 24 h. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (E1910, Promega, USA) according to the manufacturer’s instructions. Briefly, after being washed with PBS, the cells were lysed with 200 μl of the lysis buffer for each well and centrifuged to get supernatant. 30 μl supernatant was pipetted into each well of a 96-well plate and mixed immediately with firefly luciferase working solution to detect Luciferase activity. After the addition of 100 μl Renilla luciferase working solution to each well, the Renilla luciferase activity was detected. The value of luciferase activity was analyzed by a luminometer (TECAN, Sunrise, Austria). The value of relative luciferase activity (RLU) was obtained by the ratio of firefly luciferase activity to Renilla luciferase activity.

Yang X., Zhong D., Gao W., Liao Z., Chen Y., Zhang S., Zhou H., Su P, & Xu C. (2020). Conditional ablation of MAPK7 expression in chondrocytes impairs endochondral bone formation in limbs and adaptation of chondrocytes to hypoxia. Cell & Bioscience, 10, 103.

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Transient Expression of TRIM1 and HIF1α

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For transient expression in mammalian cells, full open reading frames (ORF) for TRIM1 and HIF1α were amplified using a SW480 cDNA library and inserted into the pCS2-EGFP and pCS2-Flag vectors. pRK5-HA-Ub-WT and the lysine mutants plasmids were maintained in our lab [35 (link)]. HRE-luc, pNF-κB-Luc, and pRL-TK reporter plasmids were purchased from Addgene. The sequences of all plasmids were confirmed by sequencing before use.
Antibodies for GAPDH (G9545) and Flag (F7425) were purchased from Sigma-Aldrich. EGFP (sc8334) antibody was obtained from Santa Cruz Biotechnology. Antibodies for HIF1α antibodies (D1S7W, #36169S), NF-κB p65 (D14E12, #8242), phospho-NF-κB p65 (Ser536) (93H1, #3033) and IκBα (44D4, #4812) were from Cell Signaling Technology. Anti-TRIM1/MID2 (68359-1-Ig) and anti-HA Epitope Tag (901501) antibodies were from Proteintech and Biolegend. DMOG was from Selleckchem. Cell culture products were from Invitrogen. The relevant chemicals in this study were obtained from Sigma-Aldrich unless stated otherwise.

Shi L., Fang X., Du L., Yang J., Xue J., Yue X., Xie D., Hui Y, & Meng K. (2024). An E3 ligase TRIM1 promotes colorectal cancer progression via K63-linked ubiquitination and activation of HIF1α. Oncogenesis, 13(1), 16.

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