The coding region of human USP2A (accession number NM_004205) without the stop codon was cloned into pcDNA3.2/V5-DEST (Thermo Fisher Scientific) using BP clonase II (Thermo Fisher Scientific). The resultant plasmid encoded C-terminal V5-tagged USP2A. Halo-tagged Oct-1 and Oct-2 expression plasmids were purchased from Promega (Madison, WI). The HA-tagged ubiquitin plasmid was purchased from Addgene (Cambridge, MA). The USP2A, or control pcDNA3.2/V5-DEST plasmid, plasmids encoding Halo-tagged Oct-1 or Oct-2 and HA-tagged ubiquitin were transfected into HEK293FT cells using Lipofectamine 2000 reagent (Thermo Fisher Scientific). The pull-down and elution of Oct proteins were performed using Magne HaloTag beads (Promega) and HaloTEV protease (Promega) as per the manufacturer's instructions. For detection of Western blot bands, corresponding to K48- and K63-linked ubiquitin chains, Oct proteins, HA-tagged ubiquitin, and V5-tagged USP2, 1000-fold-diluted antibodies against polyubiquitin chains formed by K48 residues (ab140601; Abcam) and K63 residues (ab179434; Abcam), Oct-1 (A301-717A; Bethyl Laboratories), Oct-2 (ab179808; Abcam), HA-tag (#3724; CST), and V5-tag (A190-120F; Bethyl Laboratories) were used as the primary antibodies.
Pcdna3.2 v5 dest
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PcDNA3.2/V5/Dest is a mammalian expression vector designed for high-level expression and purification of recombinant proteins in a variety of cell lines. It provides a V5 epitope tag for detection and purification of expressed proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Prl sv, by Promega (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Ab179808, by Abcam (1 mentions)
Ab140601, by Abcam (1 mentions)
Ab179434, by Abcam (1 mentions)
Magne halotag beads, by Promega (1 mentions)
Bp clonase 2, by Thermo Fisher Scientific (1 mentions)
Ha tagged ubiquitin plasmid, by Addgene (1 mentions)
Halotev protease, by Promega (1 mentions)
Gateway technology, by Thermo Fisher Scientific (1 mentions)
Primestar mutagenesis kit, by Takara Bio (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
In fusion cloning kit, by Takara Bio (1 mentions)
Gateway lr clonase 2 enzyme mix, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Retinoic acid, by Merck Group (1 mentions)
Transit lti, by Mirus Bio (1 mentions)
10 protocols using pcdna3.2 v5 dest
1
Investigating USP2A Interactions with Transcription Factors
2
STAT3-Dependent Luciferase Reporter Assay
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The Stat3-C Flag pRc/CMV (CA-STAT3) was a gift from Jim Darnell (Addgene plasmid #8722) [48 (link)], and pCDNA3.2/V5-DEST was from Thermo Fisher Scientific. pGL4.47[luc2P/SIE/Hygro] (a firefly luciferase reporter plasmid under the control of five copies of STAT3 responsive element) and pRL-SV (a Renilla luciferase reporter plasmid under the control of SV40 promoter) were provided by Promega. Huh7 cells in Opti-MEM medium (Thermo Fisher Scientific) were transiently transfected with pGL4.47[luc2P/SIE/Hygro] (300 ng) and pRL-SV (30 ng) for 6 h using a FuGENE® HD Transfection Reagent (Promega). After replacing the transfection medium with the growth medium containing 10% FBS, the cells were treated with 5–20 μM HsA for 18 h. In some experiments, Huh7 cells were transfected with 300 ng of CA-STAT3 in conjunction with reporter plasmids for 12 h, and then exposed to HsA. The same amount of pCDNA3.2/V5-DEST was used for mock transfection. Luciferase activities in the cell lysates were determined using a Dual-Luciferase® Reporter Assay System (Promega), and luminescence intensity of firefly luciferase was normalized by that of Renilla luciferase.
3
Gateway-Based Protein Expression Techniques
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All primer sequences and plasmids used in this study are listed in S1 Table . pEU-based expression vectors, including pEU-E01-GW, pEU-E01-bls-SrtA-GW, pEU-E01-FLAG-GW, pEU-E01-GW-FLAG, pEU-E01-AGIA-GW, and pEU-E01-GW-AGIA, were used for wheat germ cell-free protein synthesis. pcDNA3.1 and pcDNA3.2/V5-DEST (Thermo Fisher Scientific) are vectors for mammalian cell expression. p35SΩ-GW-NOST was used for protein expression in plant cells [40 (link)].
Genes of interest were subcloned using Gateway Technology (Thermo Fisher Scientific). The AGIA tag sequence was inserted at the 5′- or 3′- end of the open reading frame (ORF) by PCR. Deletion and substitution of amino acids in the AGIA sequence were conducted using the PrimeSTAR Mutagenesis Kit (Takara Bio). Human genes (DRD1, MIB2, RelA, MARCH8, OCT4, GATA3 and p53) were obtained from the Mammalian Gene Collection (MGC) full-length cDNA clone set [41 (link)]. Mouse Mdm2 was from FANTOM cDNA clones [42 (link)]. The cDNA clones of AtGID1A and AtUBQ10 were amplified from RIKEN Arabidopsis full-length cDNA (RAFL) clones [43 (link)]. Modification of expression vectors was performed using inverse PCR and In-Fusion cloning kit (Takara Bio).
Genes of interest were subcloned using Gateway Technology (Thermo Fisher Scientific). The AGIA tag sequence was inserted at the 5′- or 3′- end of the open reading frame (ORF) by PCR. Deletion and substitution of amino acids in the AGIA sequence were conducted using the PrimeSTAR Mutagenesis Kit (Takara Bio). Human genes (DRD1, MIB2, RelA, MARCH8, OCT4, GATA3 and p53) were obtained from the Mammalian Gene Collection (MGC) full-length cDNA clone set [41 (link)]. Mouse Mdm2 was from FANTOM cDNA clones [42 (link)]. The cDNA clones of AtGID1A and AtUBQ10 were amplified from RIKEN Arabidopsis full-length cDNA (RAFL) clones [43 (link)]. Modification of expression vectors was performed using inverse PCR and In-Fusion cloning kit (Takara Bio).
4
Cloning and Mutagenesis of EphA2 and Pendrin
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pCMV-Myc-pendrin was a kind gift from Professor Min Goo Lee. pcDNA3.2-V5-EphA2 construction was achieved by using GatewayTM LR ClonaseTM II Enzyme Mix (Invitrogen, 11791-020) according to the manufacturer’s instructions; pDONR223-EphA2 (Addgene, 23926) and pcDNA3.2/V5-DEST (Thermo Fisher, 12489019) were used as donor vector and destination vector. FLAG-HA-pcDNA3.1-EphA2-1-558 and FLAG-HA-pcDNA3.1-EphA2-538-976 were subclone of EphA2 intracellular domain including transmembrane domain (amino acid 1-558) and extracellular domain including transmembrane domain (amino acid 538-976) into FLAG-HA-pcDNA3.1 backbone. The primers used for domain cloning were shown in Suppementary Table 2 . Point mutagenesis of pendrin and EphA2 were generated by using KOD Hot Start Mutagenesis kit (Merck Millipore, Toyobo; 71086-3); the pcDNA3.2-V5-EphA2 and pCMV-Myc-pendrin constructs were used as backbones. The primers used for point mutagenesis were listed in Supplementary Table 2 .
5
Tau Overexpression in Differentiated SH-SY5Y
6
Recombinant Equine CD154 Protein Production
7
Constructing HCV Genotype 1 Pseudoparticles
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Plasma samples obtained from HCV infected subjects in the BBAASH cohort [15 (link),16 (link),45 ], Irish Anti-D cohort [46 (link)], and Swan Project [47 (link)] were used to construct a library of genotype 1 E1E2-expressing lentiviral pseudoparticles using a high-throughput production and screening approach. The E1E2 region was PCR amplified from cDNA reverse transcribed from viral RNA purified from subject plasma and cloned into the expression vector pcDNA3.2/V5/Dest (Invitrogen) using Gateway technology in a one-tube BP/LR reaction, as previously described [19 ].
8
PCR Amplification and Cloning of E1E2 Region
9
TaRXR Transcript Sequence Amplification
10
Drosophila Mitochondrial Dynamics Protocols
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mtYFP (pEYFP-mitochondria [mito]) and ER-YFP (pEYFP-ER) were purchased from Takara Bio, Inc. Mitochondrial targeted DsRED (mtRFP) was a gift from T. Pozzan (Venetian Institute of Molecular Medicine, Padua, Italy; Cipolat et al., 2004 (link)). pcB6-Myc-Mfn1 and pcB6-Myc-Mfn2 were a gift from M. Rojo (Salpetriere Hospital, Paris, France; de Brito and Scorrano, 2008 (link)).
Total RNA from adult Drosophila heads was purified with TRIZOL (Invitrogen). The Marf full-length cDNA was obtained by RT-PCR performed on total Drosophila head RNA. The full-length cDNA of Marf was first cloned in pDONR221 (Invitrogen) and then into pcDNA3.2/V5-DEST by Gateway cloning (Invitrogen). pAc5.1-Atl™-GFP was generated by cloning the 120 amino acids of the transmembrane domain of Drosophila atlastin (Orso et al., 2009 (link)) into pAc5.1/V5-His (Invitrogen), previously modified with the insertion of the EGFP sequence from the pEGFP-N1 vector (Takara Bio Inc.). mtGFP was generated by subcloning the cDNA from pEGFP-mito (Takara Bio Inc.) into pActin-PPA. For generation of double-stranded RNA (dsRNA)–resistant mutant of Marf (Marf-RNAiR). Invariant-coding mutagenesis A → T84 and A → T90 of pAc5.1/V5-His-Marf was performed by using the site-directed mutagenesis kit (QuikChange; Agilent Technologies).
Total RNA from adult Drosophila heads was purified with TRIZOL (Invitrogen). The Marf full-length cDNA was obtained by RT-PCR performed on total Drosophila head RNA. The full-length cDNA of Marf was first cloned in pDONR221 (Invitrogen) and then into pcDNA3.2/V5-DEST by Gateway cloning (Invitrogen). pAc5.1-Atl™-GFP was generated by cloning the 120 amino acids of the transmembrane domain of Drosophila atlastin (Orso et al., 2009 (link)) into pAc5.1/V5-His (Invitrogen), previously modified with the insertion of the EGFP sequence from the pEGFP-N1 vector (Takara Bio Inc.). mtGFP was generated by subcloning the cDNA from pEGFP-mito (Takara Bio Inc.) into pActin-PPA. For generation of double-stranded RNA (dsRNA)–resistant mutant of Marf (Marf-RNAiR). Invariant-coding mutagenesis A → T84 and A → T90 of pAc5.1/V5-His-Marf was performed by using the site-directed mutagenesis kit (QuikChange; Agilent Technologies).
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