Ha hif1alpha pcdna3

Manufactured by Addgene

HA-HIF1alpha-pcDNA3 is a plasmid vector that expresses the hypoxia-inducible factor 1-alpha (HIF1-alpha) gene with an N-terminal hemagglutinin (HA) tag. The vector is designed for eukaryotic expression.

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PcDNA3.1 (119 citations) HA-HIF1alpha P402A/P564A-pcDNA3 (4 citations) PcDNA3-HA (3 citations)

3 protocols using ha hif1alpha pcdna3

Isolation and Purification of TF3 Monomer

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TF3 monomer (purity: 92.4%) was isolated and purified using previously established method (15 (link)). Wortmannin and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) were purchased from Sigma and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Antibodies against phospho-Akt (Ser473) (p-Akt), Akt, phospho-p70S6 kinase (Thr421/Ser424) (p-p70S6K), p70S6K, eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), HIF-1α, Notch-1, c-Jun N-terminal kinases (JNK), p38 and phosphor-Forkhead Box O1 (Thr24) (p-FoxO1) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against phosphor-mammalian target of rapamycin (p-mTOR) (Ser2448), mTOR were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against phosphor-4E-BP1 (Ser65/Thr70) (p-4E-BP1), c-Myc, phosphor-extracellular signal-regulated kinases 1/2 (Thr202/Tyr204) (p-ERK1/2), ERK1/2 and GAPDH were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz). Plasmids (myrAkt delta4-129, pcDNA3-Flag mTOR wt, pWZL Neo Myr Flag RPS6KB1, pET14b PHAS-I, HA-HIF1alpha-pcDNA3, 3XFlagNICD1, pMXs-hc-Myc, ODD-Luciferase-pcDNA3, VEGF promoter, cMyc promoter (TBE1/2-wt), and pCBFRE-luc) were purchased from Addgene (Cambridge, MA, USA).

GAO Y., RANKIN G.O., TU Y, & CHEN Y.C. (2015). Theaflavin-3, 3′-digallate decreases human ovarian carcinoma OVCAR-3 cell-induced angiogenesis via Akt and Notch-1 pathways, not via MAPK pathways. International Journal of Oncology, 48(1), 281-292.

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Plasmids Transfection in HEK293T and Hela Cells

Cited 2 times

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Plasmids transfection was performed by Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher) following the manufacturer's instructions in HEK293T cells and Hela cells. HA-HIF1alpha-pcDNA3 (HA-HIF1α in short, Addgene plasmid #18949; http://n2t.net/addgene:18949; RRID: Addgene_18949), and HA-HIF1alpha P402A/P564A-pcDNA3 (Addgene plasmid # 18955; http://n2t.net/addgene:18955; RRID: Addgene_18955) were gifts from William Kaelin 24 (link). HIF1alpha(401delta603)_R27G (HA-ΔODD-HIF1α in short) was a gift from Eric Huang (Addgene plasmid #52215; http://n2t.net/addgene:52215; RRID: Addgene_52215) 25 (link). HRE-luciferase (HRE-Luc in short) was a gift from Navdeep Chandel (Addgene plasmid #26731; http://n2t.net/addgene:26731; RRID: Addgene_26731) 26 (link). Plasmid pcDNA3.1 Flag-HIF1B (#930) was a gift from James Brugarolas (Addgene plasmid #99916; http://n2t.net/addgene:99916; RRID: Addgene_99916) 27 (link). Human PPARD was initially amplified from cDNA template and then cloned into p3XFLAG-CMV™-10 (Sigma, E7658) fused with Flag or HA tag. To generate the truncated domains of PPARD (Flag-DBD and Flag-LBD) as previously described 28 (link), relevant fragments were amplified by indicated primers listed in Table S1 and then all were cloned into p3XFLAG-CMV™-10.

Wu Y., Tang X., Lee S., Hong H., Cao X., Lau C.W., Liu B., Chawla A., Ma R.C., Huang Y., Lui K.O, & Tian X.Y. (2022). Endothelial PPARδ facilitates the post-ischemic vascular repair through interaction with HIF1α. Theranostics, 12(4), 1855-1869.

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Molecular Tools for Hypoxia Signaling Analysis

Cited 1 time

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RIP140-expressing vectors (pEFcmyc-RIP140 [47 (link)], pEGFP-RIP140[48 (link)]) and control vectors, pEGFP (Clontech), are described elsewhere. HA-HIF1alpha-pcDNA3 (Addgene plasmid # 18949) and HA-HIF2alpha-pcDNA3 (Addgene plasmid # 18950) plasmids were gifts from Dr Kaelin [49 (link)]. The GLUT3-Luc reporter gene was a gifts from Dr. Yuan [50 (link)]. 2-Deoxy-d-glucose (2DG, D6134)), 6-aminonicotinamide (6AN; # A68203), 3-bromopyruvate (BrP; #16490), MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (98%, CAS 298-93-1), deferoxamine mesylate salt (D9533), di(N-succinimidyl)glutarate (80424), crystal violet (C0775), anti-Mouse IgG-FITC antibody (F6257), monoclonal Anti-β-actin-peroxidase antibody (A3854) were purchased from Sigma-Aldrich (Merck, Darmstadt, Germany). Puromycin (ant-pr-1) was purchased from Invivogen (France). The ATP Determination Kit (10700345) was from Fisher Scientific (France). Deoxy-d-glucose, 2-[1,2-3H (N)] (NET328A250UC) was from Perkin Elmer (France). GLUT3 shRNA (m) (sc-41219-V) were purchased from Santa Cruz Biotechnology (Dallas, USA). Rabbit polyclonal to RIP140 (ab42126) was from Abcam (Cambridge, UK). BrdU Hu-purified-clone B44 (#347580) was from Becton Dickinson (France). Ambion™ Silencer™ Pre-Designed siRNA specific of human GLUT3 (SLC2A3) was purchased from Fisher Scientific (#10446914, Illkirch, France).

Jacquier V., Gitenay D., Fritsch S., Bonnet S., Győrffy B., Jalaguier S., Linares L.K., Cavaillès V, & Teyssier C. (2022). RIP140 inhibits glycolysis-dependent proliferation of breast cancer cells by regulating GLUT3 expression through transcriptional crosstalk between hypoxia induced factor and p53. Cellular and Molecular Life Sciences, 79(5), 270.

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