The following expression vectors were used in the present study: p4xCSL-firefly luciferase (Addgene #41726), pCAGGS-NICD (Addgene #26891), DN-dynamin 1 (Dynamin 1 K44A, a gift from Dr. Sandra Schmid), DN-dynamin 2 (GFP-dynamin 2 K44A, Addgene #22301), HA-Ubiquitin (Addgene #18712), pHR_SFFV_LaG17_synNotch_TetRVP64 (Addgene #79128), pHR_EGFPligand (Addgene #79129), pCS2-Notch1 FL-6MT (Addgene #41728), pCMV-mouse Dll1 (OriGene), pCMV-mouse Dll4 (OriGene), p6872 pHAGE-N-V5-MAML1-FL (Addgene #37048), and pHes1-GFPd2 (Addgene #14808). pCS2-Notch1 L468A was constructed by site-directed mutagenesis using the PrimeSTAR Mutagenesis Basal Kit (Takara). DN-MAML1 lacking the NICD-binding domain (13L-74H) and Dll1ΔC and Dll4ΔC lacking the intracellular domain (Dll1:569V-728L, Dll4:552A-686V) were amplified by polymerase chain reaction (PCR) and subcloned into pCAG-RB using the In-Fusion HD Cloning Kit (Clontech). pCMV-HA-Notch1 and pCMV-Myc-Ubiquitin were constructed by subcloning the cDNAs of Notch1 or Ubiquitin into pCMV-HA-N or pCMV-Myc-N (Clontech), respectively. Mouse and chick Psd1 cDNAs (ENSMUSG00000023452 and ENSGALT00000011126.6, respectively) were chemically synthesized and subcloned into the pGL3-promoter or pCMV-Myc-N vector.
Pcaggs nicd
Manufactured by Addgene
The PCAGGS-NICD is a plasmid construct that contains the Notch Intracellular Domain (NICD) sequence. The NICD is a key regulatory domain of the Notch signaling pathway, which plays a crucial role in cellular differentiation and development. This plasmid can be used in research applications involving the study of Notch signaling and its associated cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Pcmv myc n, by Takara Bio (1 mentions)
Ha ubiquitin, by Addgene (1 mentions)
Pcmv ha n, by Takara Bio (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Phr sffv lag17 synnotch tetrvp64, by Addgene (1 mentions)
P4xcsl firefly luciferase, by Addgene (1 mentions)
Primestar mutagenesis basal kit, by Takara Bio (1 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions)
Biolux gaussia luciferase assay kit, by New England Biolabs (1 mentions)
Pgluc basic, by New England Biolabs (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Sirna transfection reagent, by Santa Cruz Biotechnology (1 mentions)
Fast green, by Merck Group (1 mentions)
Pcag gfp, by Addgene (1 mentions)
Pcagen sbp dicer1, by Addgene (1 mentions)
4 protocols using pcaggs nicd
1
Notch Signaling Pathway Expression Vectors
2
Gaussia Luciferase Reporter Construct
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To create the pGLuc-4XCSL reporter construct, the 4XCSL sites were subcloned from the pGL2-4XCSL-luciferase vector (a gift from Raphael Kopan; Addgene plasmid #41726) [32 (link)], into pGLuc-basic (New England Biolabs) using EcoRI and HindIII. The pCAGGS-NICD expression vector was a gift from Nicholas Gaiano (Addgene plasmid #26891) [33 (link)]. SW480 and HCT116 cells were seeded in technical triplicate into 6-well dishes at a density of 4 × 105 cells/well. Approximately 12–16 h post-seeding, cells were co-transfected with 1.0 μg pGLuc-4XCSL, 0.5 μg pRS-β-galactosidase, 0.175 μg pCAGGS-NICD, and the indicated amount of pcDNA3-hKaiso using TurboFect™ Transfection Reagent (Thermo Fisher cat. #R0532). Twenty-four hours post-transfection, culture media was assayed for luciferase using the Biolux™ Gaussia Luciferase Assay Kit (New England Biolabs; cat. #E3300L), and read on an LB luminometer (Thermo Fisher). Luciferase activity was normalized to β-galactosidase to control for transfection efficiency. The average luciferase values from 3 biological replicates were calculated and plotted as relative fold change against the empty pGLuc-basic vector. Statistical significance was calculated using one-way analysis of variance (ANOVA).
3
Notch-1 Knockdown and Overexpression
4
In Utero Electroporation of Mouse Embryos
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IUE was performed according to a previous publication.28 Plasmids of pCAGEN‐SBP‐DICER1 (#50558), pCAGGS‐NICD (#26891) and pCAG‐GFP (#11150) were purchased from addgene. Mouse Jag1 was amplified and cloned into HindIII/BamHI sites of p3xFLAG‐CMT‐14 vector. Pregnant mice at E13.5 were anaesthetized with isoflurane (3% for induction and 2% during surgery for maintenance). A 1.5 cm incision was made along the linea alba in the lower abdomen, and the uterine horns were exposed. Desired plasmids (1.5 mg/mL) diluted in 1ul sterile Tris‐EDTA buffer (pH 7.4), which contained Fast Green (Sigma), were injected into the lateral ventricle of embryos at E13.5. Five 50 ms pulses of 33 V with 950 ms intervals were applied with a BTX electroporation system (ECM830). After electroporation, the uterine horns were placed back and the incision was sutured. The embryonic brains were used for further experiments at E15.5.
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