pEGFP-n1-APP was created by PCR amplification of APP695 from cDNA extracted from HeLa cells and cloned into pEGFP-n1 (Clontech), followed by subcloning into pmCherry-n1 and pYFP-n1 using NheI and SalI. The intracellular domain was removed by PCR amplification of APP and cloning into pYFP-n1 using NheI and SalI. AICD and the various AICD truncation mutations were PCR amplified using pEGFP-n1-APP as template and cloned into pEGFP-n1 or pYFP-n1. For bacterial expression the various truncations were PCR amplified and cloned into a pET28 derived plasmid described in [35 (link)] using BamHI and XhoI. pEGFP-c3-2xML1N was kindly provided by Dr. H. Xu (University of Michigan) [36 (link)] and was PCR cloned into pmCherry-c1 using XhoI and BamHI. The expression plasmid for production of His-tagged Vac14 was kindly provided by Dr. L. Weisman (University of Michigan). Expression plasmids for the APLP1 and 2 intracellular domains as well as AICD mutations in the YENPTY motif were synthesised by GeneArt (Life Technologies) with codon-optimisation for E. coli and subcloned into pET28-MBP-TEV using XhoI/BamHI for bacterial expression.
Pegfp n1
Manufactured by Takara Bio
Sourced in United States, France, Japan, Germany, Canada, China
PEGFP-N1 is a mammalian expression vector that contains the enhanced green fluorescent protein (EGFP) gene. It is designed for the expression and detection of EGFP fusion proteins in a variety of cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (47 mentions)
Pcdna3, by Thermo Fisher Scientific (32 mentions)
Pmcherry n1, by Takara Bio (23 mentions)
Pegfp c1, by Takara Bio (23 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (15 mentions)
Pmcherry c1, by Takara Bio (10 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (8 mentions)
Opti mem, by Thermo Fisher Scientific (7 mentions)
Pcmv myc, by Takara Bio (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Peyfp n1, by Takara Bio (6 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (6 mentions)
Geneart, by Thermo Fisher Scientific (6 mentions)
Pegfp n1, by Thermo Fisher Scientific (6 mentions)
Fugene hd, by Promega (6 mentions)
Pgem t easy, by Promega (5 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (5 mentions)
Hispeed plasmid maxi kit, by Qiagen (5 mentions)
In fusion hd cloning kit, by Takara Bio (4 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (4 mentions)
560 protocols using pegfp n1
1
Recombinant APP Protein Expression
2
Generating SLC38A2 Mutant Constructs
3
Construction of pAd55-dE3-EGFP Recombinant Plasmid
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The plasmid pAd55-dE3-EGFP was constructed as shown in Figure 1 . First, the homologous regions E3L/E3R to the E3 gene were amplified with primers E3L1000-F/E3L1000-R and E3R1000-F/E3R1000-R, and ligated by Gibson DNA ligase into pEGFP-N1 (Clonetech, Moutain View, USA) to construct the shuttle vector pEGFP-N1-E3LR containing the homology arms. After Avr II digestion, the plasmid pAd55-FL was recovered from the agarose gel, and then transformed into BJ5183 chemically competent cells (TAKARA, China) with Mlu I-digested shuttle plasmid pEGFP-E3LR to construct plasmid pAd55-dE3-EGFP. The EGFP was introduced into the plasmid in place of the whole E3 gene by homologous recombination between E3L and E3R fragments. The recombinant plasmid was selected by colony PCR using insert-specific primers E3D-Det-F2 and AVRII-R and then verified by Ned I restriction enzyme analysis and DNA sequencing.
4
Canine CD117 and SCF Construct Generation
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The sequence of canine CD117 and SCF was synthesized by GeneArt (Thermo Fisher Scientific, Melbourne, Australia). The CD117 sequence included a secretion signal and extracellular and transmembrane domains (NCBI Reference: NP_001003181; residues 1 to 545). This was cloned in-frame with GFP into the mammalian expression vector pEGFP-N1 (TaKaRa, Mountain View, CA, USA, discontinued), using restriction sites NheI and BamHI to create the vector pEGFP-N1-CD117-GFP. The canine SCF sequence included a secretion signal, the soluble part of the extracellular domain (NCBI Reference: NP_001012753.1; residues 1 to 191), and a 6× His-tag. This was cloned into the mammalian expression vector pcDNA3.1 (+) (Thermo Fisher Scientific), using restriction sites HindIII and XhoI to create the vector pcDNA3.1 (+)-canine-SCF. DNA for transfection was prepared using a PureLink HiPure Plasmid Maxiprep kit (Thermo Fisher Scientific). A control plasmid, pEGFP-N1-CD83-GFP, containing CD83 in place of CD117, was also prepared as described previously [18 (link)].
5
Cloning and Overexpression of Arpc5 in Mouse
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We cloned the total coding sequence (CDS) of the Arpc5 gene via PCR using mouse brain tissue cDNA as the template [23 (link)]. Next, we
constructed an Arpc5 overexpression vector by linking the total coding sequence of the Arpc5 to a pEGFP-N1 vector (Clontech, Palo Alto, CA, USA). The empty
vector pEGFP-N1 was used as a control [24 (link)]. The forward and reverse primers used were 5′-CCCTCGAGATGTCGAAGAACACGGTGTC-3′ (XhoI) and
5′-CGGGATCCCTACACGGTTTTCCTTGCAG-3′ (BamHI). XhoI and BamHI were used to digest the Arpc5 cDNA fragment and the pEGFP-N1
vector, respectively, followed by ligation with T4 DNA ligase (Takara) to construct the pEGFP-N1-Arpc5 eukaryotic expression vector. We then transfected the
pEGFP-N1-Arpc5 DNA into Escherichiacoli DH5ɑ cells, and cultured them in an Luria-Bertani (LB) culture plate overnight. Single white
colonies were collected and incubated in liquid LB culture medium overnight on a shaking table. Finally, XhoI and BamHI were used to digest the recombinant
extraction plasmid pEGFP-N1-Arpc5 to confirm its integration in the correct orientation with correct sequence, which was also detected using automated DNA sequencing.
constructed an Arpc5 overexpression vector by linking the total coding sequence of the Arpc5 to a pEGFP-N1 vector (Clontech, Palo Alto, CA, USA). The empty
vector pEGFP-N1 was used as a control [24 (link)]. The forward and reverse primers used were 5′-CCCTCGAGATGTCGAAGAACACGGTGTC-3′ (XhoI) and
5′-CGGGATCCCTACACGGTTTTCCTTGCAG-3′ (BamHI). XhoI and BamHI were used to digest the Arpc5 cDNA fragment and the pEGFP-N1
vector, respectively, followed by ligation with T4 DNA ligase (Takara) to construct the pEGFP-N1-Arpc5 eukaryotic expression vector. We then transfected the
pEGFP-N1-Arpc5 DNA into Escherichiacoli DH5ɑ cells, and cultured them in an Luria-Bertani (LB) culture plate overnight. Single white
colonies were collected and incubated in liquid LB culture medium overnight on a shaking table. Finally, XhoI and BamHI were used to digest the recombinant
extraction plasmid pEGFP-N1-Arpc5 to confirm its integration in the correct orientation with correct sequence, which was also detected using automated DNA sequencing.
6
Cloning of S. frugiperda Cortactin
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To amplify the S. frugiperda cortactin gene, total mRNA was isolated from Sf21 cells using an RNeasy kit (Qiagen; 74004) and reverse-transcribed to cDNA using a Protoscript II First Strand DNA Synthesis kit (New England Biolabs; E6560S) using random hexamers as primers. S. frugiperda cortactin-specific primers were used to PCR-amplify a 1.9 kb fragment from cDNA that was then used as a template for PCR amplification with a second primer set. Next, we constructed a plasmid vector for an N-terminal GFP-tagged S. frugiperda cortactin. eGFP was PCR-amplified from pEGFP-N1 (Takara Bio USA) and inserted using Gibson assembly into a NotI/BamHI-digested pACT. The amplified cortactin fragment was then subcloned into the NotI site of the resulting plasmid using Gibson assembly. The resulting plasmid expresses GFP fused to the N-terminus of S. frugiperda cortactin (GFP-cortactin). To express a fusion of EGFP to the C-terminus of the p21 (ARPC3) subunit of the Arp2/3 complex (p21-EGFP), the T. ni arpc3 gene from pIZ-p21-EYFP (Goley et al., 2006 (link)) was PCR-amplified and subcloned using Gibson assembly, along with eGFP PCR-amplified from pEGFP-N1 (Takara Bio USA), into the BamHI site of pACT. In all cases, DNA ready for transfection was purified as described above.
7
Constructing Mutant IR Plasmid
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The ACP sequence from the ACP-GPI plasmid [25] was incorporated to the HIR plasmid previously described [24] by the Gibson assembly (New England Biolabs). The HIR (Homo sapiens, NCBI Reference Sequence: NM_001079817) with the HindIII site before the start codon and the XbaI site after the stop codon, was subcloned by replacing the EGFP sequence of pEGFP-N1 (Clontech, Takara) [24] . The sequences of the HIR, the backbone plasmid, pEGFP-N1 lacking the EGFP sequence, and the ACP were amplified by PCR with PrimeSTAR Max DNA Polymerase (Takara). The primers for PCR were listed in the S1 Table . The KD 2031-ACP-IR plasmid was generated by a point mutation at Lys 1045 to Arg (AAG to AGG).
The fragment sequence of HIR, the XhoI site at 3020 to the stop codon, in the plasmid of WT 2031-ACP-IR was replaced to the XhoI-XbaI fragment of HIR sequence including the K1045R mutation with the XbaI site after the stop codon synthesized by GeneArt (ThermoFisher). The sequences of plasmids were verified and in the case of plasmids generated by Gibson assembly all sequences including the plasmid backbone were verified by sequencing by Fasteris SA.
The fragment sequence of HIR, the XhoI site at 3020 to the stop codon, in the plasmid of WT 2031-ACP-IR was replaced to the XhoI-XbaI fragment of HIR sequence including the K1045R mutation with the XbaI site after the stop codon synthesized by GeneArt (ThermoFisher). The sequences of plasmids were verified and in the case of plasmids generated by Gibson assembly all sequences including the plasmid backbone were verified by sequencing by Fasteris SA.
8
Plasmid Constructs for Immune Receptor Studies
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pCMV6-XL4-CD32A (Cat. No. SC112914), pCMV6-XL5-CD32B (Cat. No. SC128159), pCMV6-XL5-CD32C (Cat. No. SC124933), pCMV-CD64A-GFP (Cat. No. RG207487) and pCMV-CD16A-GFP (Cat. No. RG219204) were purchase from Origene. pBK-CMV-FynN18-GFP (encoding only the first 18 amino acid of the N-terminal part (membrane domain) and pBK-CMV-LckN18-GFP (encoding only the first 18 amino acid of the N-terminal part (membrane domain) have been reported.85 (link) pcDNA3.1 (+T7) human CXCR7 and pcDNA3.1 (+T7) human CXCR4 were kindly provided by J. Bernhagen, LMU München. To clone plasmids encoding GFP fusion proteins, cDNAs for human CD32A, CD32B, CD32C, CCR5, H2B and SAMHD1 were amplified by PCR and inserted in pEGFP-N1 (Clontech) using AgeI and EcoRI. For site-specific mutagenesis, two overlapping primers carrying the mutations were used to amplify the plasmids followed by DpnI digestion. To clone mtagBFP fusion proteins for human CD32A, CD32B, CD32C, and the corresponding mutated isoforms, the receptor encoding insert was cloned from the corresponding GFP fusion protein plasmids by restriction digest with AgeI and EcoRI pCMV-mtag BFP. For cloning of pCCR5-GFP, human CCR5 was amplified by PCR and inserted in pEGFP-N1 (Clontech) using SalI and NheI. All oligos used for amplification are listed in the Table S1 .
9
Fluorescent Protein Tagging of Mitotic Proteins
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An mCherry-Ndc80 plasmid (pMC299) was constructed by sub-cloning Ndc80 cDNA from pEGFP-Hec1 (kind gift of Eric Nigg) using KpnI and HindIII. Ndc80 cDNA was then amplified and ligated into tagRFP-N1 (pMC387; pEGFP-N1 (Clontech, Mountain View, CA) in which eGFP was replaced by tagRFP), creating Ndc80-tagRFP (pMC389). A Bub3-eGFP plasmid (pMC360) was generated by amplification by PCR of the short isoform of Bub3 cDNA using forward primer 5’-GCGCTCGAGATGACCGGTTCTAAC-3’ and reverse primer 5’-CGCCTGCAGAGTACATGGTGACTT-3’, and ligated into pEGFP-N1 (Clontech) using XhoI and PstI. To generate a HaloTag-CENP-A plasmid (pMC442), full-length CENP-A cDNA (GeneArt #2015ABQWHP, Thermofisher, Waltham, MA) was inserted into a pHTN HaloTag CMV-neo plasmid (Promega, Madison, WI) using EcoRI and XbaI sites. All constructs were confirmed by sequencing (Source BioScience, UK).
10
Recombinant Gag-GFP Constructs for Yeast and Mammalian Expression
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The templates for Gag-GFP and Gag(p6T*)-GFP coding sequence amplification (pGag-EGFP, pGagLTAL-EGFP) were gifts of Marylin Resh (130 (link)). We amplified the coding sequences by PCR with primers containing appropriate restriction sites and constructed the mutant Gag-GFP versions by PCR mutagenesis. For expression in yeast, we subcloned the coding sequences into pYPGE2 (PGK promoter, CYC1 termination signal) or pRS425 and pRS415 with MET3 promoter and CYC1 termination signal. The MET3 promoter and CYC1 termination signal sequences were as described (131 (link)). To gain sufficient Gag-GFP expression from pEGFP-N1 (Clontech) in HEK293 cells, we subcloned 38 bp upstream of the ATG in pGag-EGFP in front of the ATG in pEGFP-N1. To express GST-tagged Gag domains in E. coli, we used pGEX-6-P1. All constructs were verified by sequencing. E. coli strains XL1-blue and DH5 α were used for cloning. We purchased plasmids for the expression of epitope-tagged ALIX and TSG101 from Gene Copoeia (pReceiver vector).
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