The human HTT-exon1 constructs (Q72 and Q25) were synthesized by Thermo Fisher Scientific and subcloned into the pcDNA/DEST40 vector (Thermo Fisher Scientific, #12274-015). The mouse full-length Hipk3 cDNA was codon-optimized and synthesized by Thermo Fisher Scientific and subcloned into the pTT5SH8Q2 vector (NRC Biotechnology) with N-terminal GFP tag. The human or rat Hipk3 cDNA failed to express efficiently in STHdh cells. The Q72-GFP construct (expressing Methionine-Q72-GFP) was cloned by PCR amplification from the HTT-exon1 constructs, and ligation to the pcDNA-GFP plasmid. The QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) was used to generate the point mutation constructs: kinase-dead mouse Hipk3 (K226M and D322N), DN-MAPK11 (TGY (180-182) to AGF). The MAPK11 (#20355) and GFP-LC3B (#24987) construct was obtained from Addgene.
Pcdna dest40 vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PcDNA-DEST40 vector is a plasmid vector designed for use in eukaryotic expression systems. It is a Gateway-compatible vector that enables simple and efficient directional cloning of blunt-end PCR products into the vector.
Automatically generated - may contain errors
Lab products found in correlation
Lr clonase 2 reaction, by Thermo Fisher Scientific (2 mentions)
Gfp lc3b, by Addgene (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Lr clonase enzyme, by Thermo Fisher Scientific (1 mentions)
Hispeed plasmid purification midi kit, by Qiagen (1 mentions)
Pentry gfp, by Addgene (1 mentions)
One shot top10 chemically competent e coli, by Thermo Fisher Scientific (1 mentions)
Jetprime, by Polyplus Transfection (1 mentions)
V5 tag, by Thermo Fisher Scientific (1 mentions)
Geneart site directed mutagenesis system, by Thermo Fisher Scientific (1 mentions)
Pegfp c3 vector, by Takara Bio (1 mentions)
Pmscv mcherry syk, by Addgene (1 mentions)
Pcmv5 shp2, by Addgene (1 mentions)
Pcmv5 src, by Addgene (1 mentions)
Pcgn ha n ras, by Addgene (1 mentions)
Gateway cloning technology, by Thermo Fisher Scientific (1 mentions)
Gateway system, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
11 protocols using pcdna dest40 vector
1
Huntingtin and Hipk3 Constructs in Cell Line
2
Generating Plasmids and Reporter Constructs
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Plasmids pCI-λNHA-MARF1 variant plasmids were generated by conventional molecular cloning techniques using MfeI and NotI restriction enzyme sites. Point and deletion mutations within the coding sequence of MARF1 were made through site-directed mutagenesis with Phusion Hot-Start II DNA polymerase. Similarly, Renilla luciferase (RL) reporter plasmids containing gene-specific 3’UTR regions were generated through conventional cloning using XbaI and NotI restriction enzyme sites. The RL-5BoxB and firefly luciferase (FL) plasmids have been previously described (Nishimura et al., 2018 (link)). V5-tagged EDC4 was generated by Gateway cloning pDONR-EDC4 into pcDNA-DEST40 vector (thermo).
3
Characterization of DDX60 and IRES Constructs
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DDX60 wild‐type, IRF1, and Fluc plasmid DNA was generated by (Schoggins et al, 2011 (link)) in pTRIP.CMV.IVSb.ires.TagRFP‐DEST backbone (GenBank OM859267, OM859269, and OM859268, respectively). Empty vector is pTRIP.CMV.IVSb.ires.TagRFP‐DEST without ccdB suicide gene and Fluc negative control plasmid has Fluc sequence in place of an ISG. DDX60 wild‐type, IRF1, and Fluc DNA was also cloned into a pTRIP.CMV.IVSb.ires.TagRFP‐DEST backbone containing a puromycin selectable marker (pTRIP.CMV.IVSb.ires.TagRFP‐puro‐DEST). GFP plasmid is EGFP cloned into pcDNA DEST40 vector (Thermo Fisher Scientific 12274015). RIG‐I plasmid DNA was generated by (Dittmann et al, 2015 (link)) in pSCRPSY lentiviral expression vector. Mutant DDX60 constructs and bicistronic 5′ cap‐driven Rluc and IRES‐driven Fluc constructs (Honda et al, 2000 (link)) can be found using GenBank accession numbers: DDX60 A785V: OM859270, DDX60 K791N: OM859271, DDX60 E890A: OM859272, DDX60 S918/T920/A: OM859273, DDX60 Q1321A: OM859274, DDX60 R1328A: OM859275, DDX60 ∆1,402–1,712: OM859276, DDX60 ∆1–556: OM859277, DDX60 ∆1–428: OM859278, Polio IRES: OM859279, EV71 IRES: OM859280, EMCV IRES: OM859281, FMDV IRES: OM859282, HCV IRES: OM859283, BVDV IRES: OM859284.
4
Overexpression of Human PPM1A
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Wild-type human PPM1A (Hu-PPM1A) cDNA was cloned into the pcDNA-DEST40 vector (Invitrogen, San Diego, CA) according to the manufacturer's recommendations. All transfections were carried out using the Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, USA).
5
Overexpression of Sphingosine Kinase-1 in SCC-25 Cells
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LR clonase enzyme (Invitrogen) was used to insert entry vector, purified pDONR223-SPHK1 (human Sphingosine Kinase-1, plasmid 23704, Addgene, Cambridge MA), into destination vector pcDNA-DEST40 Vector (12274-015, Invitrogen). One Shot TOP10 Chemically Competent E. coli (C4040-03, Invitrogen) was transformed with SphK1-DEST-40 plasmid DNA. A single colony was recovered and propagated overnight and the plasmid was purified using a HiSpeed Plasmid Purification Midi Kit (Qiagen, Valencia, CA).
SCC-25 cells were then transfected using jetPRIME (PolyPlus transfection, New York) according to manufacturer’s guidelines. Briefly, 4 μl of jetPRIME solution was added to 2 μg of DNA in jetPRIME buffer and incubated for 10 minutes at room temperature. The transfection solution was added to cells at 60-80% confluency; transfection media was removed 4 hour post-transfection. Geneticin (G418, Life Technologies, Carlsbad, CA) selection began 2–3 days later. SphK1 mRNA level was measured to confirm successful transfection. Green fluorescence protein (GFP) was used as a mock vector (pENTRY-GFP, Plasmid 15301, Addgene).
SCC-25 cells were then transfected using jetPRIME (PolyPlus transfection, New York) according to manufacturer’s guidelines. Briefly, 4 μl of jetPRIME solution was added to 2 μg of DNA in jetPRIME buffer and incubated for 10 minutes at room temperature. The transfection solution was added to cells at 60-80% confluency; transfection media was removed 4 hour post-transfection. Geneticin (G418, Life Technologies, Carlsbad, CA) selection began 2–3 days later. SphK1 mRNA level was measured to confirm successful transfection. Green fluorescence protein (GFP) was used as a mock vector (pENTRY-GFP, Plasmid 15301, Addgene).
6
Cloning and Tagging of Protein Constructs
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Wild-type MLH1, HSPA8, and BRIP1 entry clones are from the human ORFeome v8.1 collection [61] (link). Using Gateway LR reactions, wild-type MLH1, mutant MLH1 (I107R), and GFP were transferred into the pMSCV-N-FLAG-HA-PURO vector [65] (link); HSPA8 and BRIP1 were transferred into the pcDNA-DEST40 vector that contains a C-terminal V5 tag (Invitrogen 12274-015).
7
Generation and Characterization of TDP-43 Constructs
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The full-length coding region of isoform I or isoform II of human TDP-43 cDNA (Figure 1E ) was isolated from a human cDNA library (Clontech) and subcloned into a pcDNA DEST-40 vector (Invitrogen) (14 (link),18 (link)). The original plasmid encoding the minigene was provided by Dr Hurng-Yi (National Taiwan University) (19 (link)). The wild-type exon 6 of TDP-43 and the enhanced-green fluorescence protein (EGFP)-fused minigene were amplified by polymerase chain reaction (PCR) with specific primers from the cDNA library and subcloned into the original minigene construct or the pEGFP-C3 vector (Clontech). The Δintron 6 or Δintron 7 minigene, AU-rich element mutant construct and spliced site mutant constructs were produced using the GeneArt site-directed mutagenesis system (Invitrogen). The primer sequences for construction of these plasmids are listed in Supplementary Table S2.
8
Plasmid Construction and Characterization of Ras and Associated Proteins
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A plasmid encoding human pBabe-KRAS4B WT was generously provided by Channing Der (University of North Carolina, Chapel Hill), which was subcloned into pcDNA3 using KpnI and NotI to integrate an N-terminal HA tag. KRAS mutants (G12V, Y32F and Y64F) were generated by site-directed mutagenesis. Human pcDNA3-HA-HRAS, pCGN-HA-NRAS, pcDNA3-Flag-RAC2, pCDNA-HA-FAK P712/715A, pCDNA-HA-FAK Y576/577F, pMSCV-mCherry-SYK, pCMV5-Src (WT or K295RY527F), and pCMV5-SHP2 (WT, E76K or C459S) were obtained from Addgene. Flag-SHP2 constructs were subcloned into pcDNA3 and a plasmid encoding HA-CBL was subcloned into the pcDNA-DEST4.0 vector using Gateway Cloning technology (Invitrogen). Plasmids were verified by direct DNA sequencing.
9
Cloning and Expression of FUCA1 and FUCA2
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The coding sequences of H. sapiens FUCA1 (NCBI reference sequence XM_005245821.1, using forward primer 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCACCACCATGCGGGCTCCGGGGATG-3′ and reverse primer 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACTTCACTCCTGTCAGCTTTAT-3′), and of H. sapiens FUCA2 (NCBI reference sequence NM_032020.4, using forward primer 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTC CACCACCATGCGGCCCCAGGAGCTC-3′ and reverse primer 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCT TAGATCACATTAGTCAGGGCTA-3′) were amplified via PCR and cloned into pDNOR-221 and thereafter sub-cloned in pcDNA™-DEST40 vector using the Gateway system (Invitrogen). Correctness of all constructs was verified by sequencing. Confluent COS-7 cells were transfected with pcDNA3.1 empty vector (Mock) or the vector with described insert in conjunction with FuGENE (Roche). After 72 hours, medium isolated and frozen at –80 °C and cells were harvested by scraping in 25 mM potassium phosphate buffer (pH 6.5, supplemented with 0.1% (v/v) Triton X-100 and protease inhibitor cocktail (Roche)). After determination of the protein concentration (BCA kit, Pierce), lysates were aliquoted and frozen at –80 °C.
10
Cysteine Mutation and Plasmid Construction
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