Mouse KCNH6 and Munc-18-1 cDNAs were derived from MIN6 cells. Point and deletion mutants were generated using a standard PCR-based mutagenesis strategy and were verified by DNA sequencing. The sequences of the primers used are listed in Supplementary Table 2. These cDNAs were subcloned into pcDNA3-HA, pcDNA3-FLAG vector (Invitrogen) or mCherry-C1 vector (Clontech) as described previously [24 (link)]. Insulin-EGFP was generated as described previously [25 (link)]. To generate recombinant adenoviruses, KCNH6 WT and KCNH6 R246A/T248A/L250A (3A) mutant were inserted into pENTR-3C (Invitrogen) and were transferred into pAd/CMV by LR Clonase recombination (Invitrogen), which co-produces red fluorescent protein (Cherry) to allow identification of transfected cells. To express an exogenous protein, HEK293A cells were transfected with the plasmids using Lipofectamine 3000 reagent (Invitrogen), whereas MIN6 cells were infected with adenoviruses.
Pcdna3 flag vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The pcDNA3-Flag vector is a mammalian expression vector that enables the expression of recombinant proteins with a FLAG tag. The FLAG tag is a short peptide sequence that can be used for the detection and purification of the expressed proteins. The vector contains a cytomegalovirus (CMV) promoter for high-level expression in mammalian cells and an ampicillin resistance gene for selection in bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Mcherry c1 vector, by Takara Bio (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Pcdna3 ha, by Thermo Fisher Scientific (1 mentions)
Pentr3c, by Thermo Fisher Scientific (1 mentions)
Lr recombination clonase, by Thermo Fisher Scientific (1 mentions)
Pcdna3 ha rat hop, by Addgene (1 mentions)
Pcdna5 v5 human hsc70, by Addgene (1 mentions)
Pcdna3 ha human hsp90β, by Addgene (1 mentions)
Flag p21 wt, by Addgene (1 mentions)
Block it u6 rnai entry vector kit, by Thermo Fisher Scientific (1 mentions)
Pgex 4t 1, by GE Healthcare (1 mentions)
Pdsred1 n1 vector, by Takara Bio (1 mentions)
Pcdna3 vector, by Thermo Fisher Scientific (1 mentions)
Pcdna3 myc human cullin 4a 4b, by Addgene (1 mentions)
Pgv186 retroviral vector, by Genechem (1 mentions)
7 protocols using pcdna3 flag vector
1
Adenoviral Expression of KCNH6 Mutants
2
Generation of Leukodystrophy-Causing ClC-2 Mutants
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Human ClC-2 cDNA (NM_004366) was subcloned into the pcDNA3-Flag vector (Invitrogen, Carlsbad, CA, USA) to generate the N-terminal Flag-tagged human ClC-2 (Flag-hClC-2) construct. Leukodystrophy-causing ClC-2 mutations (A500V and G503R) were generated by site-directed mutagenesis, followed by verification with DNA sequencing. Other cDNA constructs employed in this study include pcDNA3.1-Myc mouse Aha1, pcDNA3.1-Myc mouse FKBP8, pFlag-CMV2 human Erg, pcDNA3-HA rat HOP, pcDNA5-V5 human Hsc70 (Addgene 19514, Watertown, MA, USA), pcDNA3-HA human Hsp90β (Addgene 22847, Watertown, MA, USA), and pcDNA3-Flag human KV4.3.
3
Cloning of Human Chk1, p21, and Other Genes
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The human Chk1 cDNA (NCBI accession number NM_001114122.2) was amplified from plasmid pF1K-Chk1 (Promega; FXC03424) and cloned into downstream of the Flag tag sequence in the pcDNA3-Flag vector (Invitrogen) to generate pcDNA3-Flag-Chk1. The human p21 cDNA (NM_001291549.1) was amplified from Flag-p21-WT (Addgene plasmid # 16240; a gift from Dr. M. C. Hung)48 (link). The cDNA fragments of human PVRL4 (NM_030916.2), PRODH (NM_016335.4), LY6D (NM_003695.2), DAO (NM_001917.4), and EPN3 (NM_017957.2) were amplified from a cDNA sample prepared from U2OS cells. The cDNA fragment of human GPR172B (NM_001104577.1) was amplified from a plasmid containing PAR2 (GPR172B) cDNA (kindly provided by Dr. Y. Takeuchi). The resultant fragments were digested with appropriate restriction enzymes and cloned into downstream of the HA tag sequence in the pcDNA3-HA vector. To generate Chk1 mutant (S345A), PCR was performed using mutagenic primers. The sequences of primers used for plasmid constructions are listed in Supplementary Table 1 .
4
Bnip3 and Bnip3Δex3 Construct Generation
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The Bnip3 gene was amplified from rat genomic DNA while Bnip3Δex3 PCR product was purified from radioactive gel. Both PCR products were cloned into pcDNA3-HA expression vector or pcDNA3-Flag vector (CMV promotor and pcDNA backbone; Invitrogen) to generate expression plasmids encoded either Bnip3 or Bnip3Δex3 HA-tag or Flag-tag constructs. ShRNA-Bnip3FL was designed to specifically target exon 3 of full-length Bnip3 (Bnip3FL), and the sequence was 5′-CACCGACACCACAAGATACCAACAGCGAACTGTTGGTATCTTGTGGTGTC-3′. shRNA-Bnip3FL was constructed with BLOCK-iT U6 RNAi entry vector kit (Invitrogen) and the control LacZ shRNA was provided in the kit. The sequence was 5′-CACCGCTACACAAATCAGCGATTTCGAAAAATCGCTGATTTGTGTAG-3′. siRNA-Bnip3Δex3 to knock down Bnip3 spliced variant (Bnip3Δex3) was designed to target the exon 2–4 junction and the sequence was 5′-CACTGTGACAGTCTGAGGATT-3′ (Gang et al., 2011 (link)); the scramble control siRNA sequence was 5′-CACCGCTACACAAATCAGCGATT-3′. The siRNA-PDK2 oligo was purchased from Invitrogen (5′-GCCTGCCTGTCTACAACAA-3′); the negative siRNA control sequence is 5′-GCCGTCCATCTAACGTCAA-3′. All the constructs were confirmed by DNA sequencing.
5
Plasmids for NF-κB Signaling Study
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Plasmids encoding Myc-CARMA1, Myc-CARMA1 truncated forms, EGFP-CARMA1, Myc-Bcl10, PKCθ WT, PKCθ AE [32] (link), IKKβ, and GFP-NF-κB RelA were kind gifts from Dr. Xin Lin. Expression vectors for FLAG-CARMA1 and HA-Bcl10 were generated by subcloning of coding sequence into pcDNA3 vector (Invitrogen). GST-CARMA1 CD-CC was generated by subcloning of coding sequence into pGEX-4T-1 (GE Healthcare, Buckinghamshire, UK). Human CKIP-1 cDNA was generated by PCR amplification from Jurkat cDNA and cloned into pcDNA3/hygro and pcDNA3-FLAG vector (Invitrogen). Expression vector for DsRed-CKIP-1 was generated by subcloning of coding sequence into pDsRed1-N1 vector (Clontech, Mountain View, USA). ΔLZ-CKIP-1 and ΔPH-CKIP-1 truncated form were generated by PCR amplification from WT CKIP-1 expression vector and subcloned into pcDNA3/hygro vector.
6
Genetic Constructs for ClC-2 and Cullins
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Mouse ClC-2 cDNA was subcloned into the pcDNA3-Flag vector (Invitrogen, Carlsbad, CA, USA) to generate the N-terminal Flag-tagged ClC-2 construct. Myc-tagged ClC-2 in the pcDNA3 vector was generated by inserting the epitope sequence between the residues V420 and E421 in the extracellular linker between helices L and M. Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15,818–15,823, Watertown, MA, USA), pcDNA3-Myc human cullin 4A/4B (Addgene 19,951, 19,922, Watertown, MA, USA), pcDNA3-HA lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19,918, Watertown, MA, USA), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taipei, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Gwangju, Korea).
7
Generation of Flag-hClock Expression Plasmid
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The full-length complementary (c)DNA of hClock (Genbank accession number, NM_004898) was amplified and inserted into the pcDNA3-Flag vector (Invitrogen Life Technologies, Carlsbad, CA, USA) in order to generate the pcDNA3-Flag-hClock expression plasmid. The cDNA fragment of Flag-hClock was then inserted into pGV186 retroviral vector (Shanghai GeneChem Co., Ltd., Shanghai, China) to generate pGV186-Flag-hClock. All the constructs were confirmed by DNA sequencing, which was performed by Shanghai GeneChem Co., Ltd.
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