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.
3xflagnicd1
Manufactured by Addgene
The 3XFlagNICD1 is a plasmid product offered by Addgene. It is a DNA construct containing a 3X FLAG-tagged version of the NICD1 protein coding sequence. The 3X FLAG tag is a protein tag commonly used for detection and purification purposes.
Automatically generated - may contain errors
Lab products found in correlation
Prl pgk, by Promega (2 mentions)
Recombinant mouse dll4, by BioLegend (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Pgl3 basic vector, by Promega (2 mentions)
Myrakt delta4 129, by Addgene (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Pcdna3 flag mtor wt, by Addgene (1 mentions)
Notch1, by Cell Signaling Technology (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
N n 3 5 difluorophenacetyl l alanyl s phenylglycine t butyl ester dapt, by Merck Group (1 mentions)
Odd luciferase pcdna3, by Addgene (1 mentions)
Pcbfre luc, by Addgene (1 mentions)
P 4e bp1, by Santa Cruz Biotechnology (1 mentions)
Pmxs hc myc, by Addgene (1 mentions)
Ha hif1alpha pcdna3, by Addgene (1 mentions)
4e bp1, by Cell Signaling Technology (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
Hif 1α, by Cell Signaling Technology (1 mentions)
Pcag mgfp, by Addgene (1 mentions)
5 protocols using 3xflagnicd1
1
Luciferase Reporter Assay for Sel1l and Hrd1 Promoters
2
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).
3
Overexpressing NICD in B16/F-10 Cells
4
Plasmid Constructs for Gene Expression
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The pEF1α-EGFP plasmid expressing nuclear EGFP has been previously reported56 (link). Glo1 and control shRNAs in the pSUPER vector were published previously20 (link). The shRNAs against human GLO1 (GATGGCTACTGGATTGAAA) were cloned into the pSUPER vector. The shRNA against mouse GAPDH was obtained from EZbiolab. To generate luciferase reporter plasmids, the 3’UTR of Notch1 mRNA was amplified from genomic DNA by PCR and cloned into the pmirGLO vector (Promega) downstream of the Firefly Luciferase gene. The AU-rich element (141–367 of 3’UTR) was deleted to generate the Notch1 3’UTR-dARE plasmid. A Renilla Luciferase, serving as the internal control, is expressed independently from the same pmirGLO plasmid. The pCAG-mGFP (#14757), CBFRE-EGFP (#17705) and 3XFlagNICD1 (#20183) plasmids were obtained from Addgene. Myc-DDK-GAPDH was obtained from the Origene. Human GLO1 cloned into lentiviral vector pLX304, from ORFeome initiative, was acquired from DNASU. All clones were verified by sequencing. Recombinant rabbit GAPDH was purchased from Sigma.
5
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.
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