The full-length cDNA of human Leupaxin (NM_004811) was cloned in frame into the EcoRI and XhoI restriction sites of the pCMV-Myc vector (Clontech, Heidelberg, Germany) to produce a Leupaxin-Myc fusion protein. Likewise, full-length cDNA of human l-Caldesmon (NM_004342) was cloned into the pEGFP-C1 vector using the XhoI and EcoRI restriction sites and into pCMV-Myc vector using the SfiI and XhoI restriction sites (both Clontech, Heidelberg, Germany) to produce EGFP-Caldesmon and Caldesmon-Myc fusion proteins, respectively. Fusion constructs were expressed in PC-3, DU 145 and LNCaP cells to analyze subcellular localization. For GST pulldown, full-length cDNA of human Leupaxin as well as LD motifs (aa 32-176) and LIM (aa 173-417) domains were cloned into the EcoRI and XhoI sites of the bacterial expression vector pGEX-4T3 (GE Healthcare, Munich, Germany) to produce GST-LPXN, GST-LPXN-LD and GST-LPXN-LIM fusion proteins.
Pcmv myc vector
Manufactured by Takara Bio
Sourced in United States, Japan
The PCMV-Myc vector is a plasmid-based expression vector designed for the efficient expression of target genes in mammalian cells. It contains a powerful cytomegalovirus (CMV) promoter to drive high-level transcription, as well as a Myc epitope tag sequence for the detection and purification of the expressed proteins.
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Lab products found in correlation
Pcmv ha vector, by Takara Bio (7 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
Pegfp c1 vector, by Takara Bio (3 mentions)
Pegfp c1, by Takara Bio (3 mentions)
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (3 mentions)
Pcmv flag vector, by Takara Bio (2 mentions)
Mg132, by Merck Group (2 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (2 mentions)
Pcmv myc, by Takara Bio (2 mentions)
In fusion hd cloning kit, by Takara Bio (2 mentions)
Pcmv ha, by Takara Bio (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dharmafect transfection reagent, by Horizon Discovery (2 mentions)
Viafect transfection reagent, by Promega (2 mentions)
Lamp1 mcherry plasmid, by Addgene (2 mentions)
Pcmv tag2b vector, by Agilent Technologies (2 mentions)
X tremegene 9, by Roche (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Lentiviral polg shrna, by Genechem (2 mentions)
70 protocols using pcmv myc vector
1
Cloning and Expression of Leupaxin and Caldesmon
2
Subcellular Localization and Co-IP of bfACP3 and bfTRAF6
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For the study of the subcellular localization and coimmunoprecipitation (Co-IP) test between bfACP3 and bfTRAF6, full-length bfACP3 was inserted into the pcDNA3.0 vector (Clontech) with a C-terminal HA Tag (transformed by our laboratory, the HA coding sequence was inserted after the XbaI restriction site) and bfTRAF6 was fused with Flag tag and inserted into the pcDNA 3.0 vector (Clontech, transformed by our laboratory, the Flag coding sequence was inserted in front of the Kpn I restriction site). For the expression of the truncated mutants of bfACP3, PCR fragments encoding amino acids 24-561, 24-204, 205-561, 205-358, 165-358, 124-358, 24-358 and 359-561 were fused with myc tag, and inserted into the expression plasmid pCMV-Myc vector (Clontech). For the reporter assays and ubiquitination experiment, full-length bfACP3 was cloned into the pCMV-Myc vector (Clontech) and bfMyD88 was constructed in the same way as bfTRAF6. The full-length sequences of bfMyD88 and bfTRAF6 are shown in the Supplementary Figures 6 7 Table 1
3
Cloning and Mutagenesis of USP4 and HDAC2
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Full-length USP4 was amplified by PCR and subcloned into pCMV-Myc vector (Clontech, Mountain View, CA, USA). The pCMV-Myc-USP4 (C311A) mutant was generated with a QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA, USA). FLAG-tagged HDAC2 was PCR amplified and cloned into pCMV-Tag2 vector (Stratagene). USP4 and HDAC2 deletion mutants were constructed by PCR and inserted into their respective expression vectors. All constructs derived from PCR products were verified by DNA sequencing.
4
Plasmids for HBV, NF-κB, and HBV Enhancer Reporters
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The plasmids for HBV 1.256 (link), NF-κB-luc57 (link), and HBV enhancer-luciferase reporters10 (link) were used in our previous reports. The plasmids for pCAGGS control, IL-32α, IL-32β58 (link), and IL-32γ59 (link) (GenBank no: BC009401) were also described previously. The expression plasmids for pHNF4α-Myc and pHNF1α-Myc were constructed by PCR amplification with a HepG2 cDNA library as a template and subcloned into the pCMV-Myc vector (Clontech, Mountain View, CA, USA).
5
Exploring FXR1 and CMAS Interactions
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For two-hybrid screening, the full open reading frame of the human FXR1 (GenBank NM_005087.3) gene was subcloned into the pGBKT7 vector (28 (link)) (Clontech Laboratories, Inc., Mountainview, CA, USA) to construct pGBKT7-FXR1 (Table I ), which was used as the bait plasmid. In the co-immunoprecipitation assay, the full open reading frames of the human FXR1 gene and human CMP-N-acetyl-neuraminic acid synthetase (CMAS) gene (GenBank NM_018686.4) were subcloned into the pCMV-HA vector (28 (link)) (Clontech Laboratories, Inc.) and pCMV-Myc vector (28 (link)) (Clontech Laboratories, Inc.) to construct the recombinant vectors, pCMV-HA-FXR1 and pCMV-Myc-CMAS. In the colocalization assay, the full open reading frame of the human FXR1 gene and human CMAS gene were subcloned into the pEGFP-N1 vector (28 (link)) (Clontech Laboratories, Inc.) and pDsRed-Monomer-N1 vector (28 (link)) (Clontech Laboratories, Inc.), respectively, to construct pEGFP-N1-FXR1 and pDsRed-Monomer-N1-CMAS (Table I ). Additionally, the full open reading frame of the human FXR1 gene was inserted into the pcDNA3.1(−) vector (29 (link)) (Clontech Laboratories, Inc.) to generate the recombinant vector, pcDNA3.1(−)-FXR1 (Table I ), which was transfected into SH-SY5Y cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and biological effect of the interaction between FXR1P and CMAS was detected.
6
HBx Truncates and Siah-1 Interaction
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pCMV-Myc-HBx was kindly provided by Dr. Minghua Zhu (Changhai Hospital, the Second Military Medical University of China). Three HBx truncates plasmids were generated with TaKaRa Mutagenesis Kit to introduce stop codon TAA into pCMV-Myc-HBx corresponding site. The human Siah-1 cDNA was subcloned into PCMV-Myc vector (Clontech) via amplifying liver marathon cDNA library of human. The heat shock response element (HSE)-luc constructs were purchased from Clontech.
Specific antibodies we used included: anti-GFP (Santa Cruz, USA), anti-Siah-1 (Abcam, USA), anti-HA (CST, USA), anti-Myc and anti-β-Actin (Sigma, USA).
Specific antibodies we used included: anti-GFP (Santa Cruz, USA), anti-Siah-1 (Abcam, USA), anti-HA (CST, USA), anti-Myc and anti-β-Actin (Sigma, USA).
7
Cloning and Expression of SYCE Proteins
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COS-7 (green monkey kidney) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS). The cells were maintained in a 95% air and 5% CO2 environment at 37°C.
Gene fragments corresponding to full-length SYCE3, SYCE3 N-helix (aa 1–52), and SYCE3 C-helix (aa 53–88) were cloned into the pEGFP-C1 vector (Clontech). The SYCE3 gene was also cloned into the pcDNA4/myc-His A vector (Invitrogen), and 7 Myc tags were inserted after the SYCE3 gene. This modified plasmid was named pcDNA4/8myc-His A. Gene fragments corresponding to full-length SYCE1 and SYCE1deltaC (aa 1–219) were cloned into the pCMV-Myc vector (Clontech).
Gene fragments corresponding to full-length SYCE3, SYCE3 N-helix (aa 1–52), and SYCE3 C-helix (aa 53–88) were cloned into the pEGFP-C1 vector (Clontech). The SYCE3 gene was also cloned into the pcDNA4/myc-His A vector (Invitrogen), and 7 Myc tags were inserted after the SYCE3 gene. This modified plasmid was named pcDNA4/8myc-His A. Gene fragments corresponding to full-length SYCE1 and SYCE1deltaC (aa 1–219) were cloned into the pCMV-Myc vector (Clontech).
8
HIF-1α Transcriptional Activity Monitoring
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The p2.1 reporter was purchased from ATCC and is commonly used for monitoring HIF-1α transcriptional activity [34 (link)]. Hypoxia response element (HRE) reporter and SOD2 promoter luciferase reporter were provided by Navdeep Chandel and Xin-Hua Feng, respectively. The HRE-reporter contains three hypoxia response element (HRE) repeats and is commonly used for monitoring HIF-1α transcriptional activity [35 (link)]. The wild-type EPEC NleB gene was originally obtained from Feng Shao, and its enzymatically dead mutant (D221/223A) was PCR amplified and subcloned into the pCS2-EGFP vector and pGEX-4T vector. Wild-type human HIF-1α was subcloned into the pCMV-Myc vector and pCMV-HA vector (Clontech). Human HIF-1α-330-399aa was cloned into the pET32a vector. All R/K mutants of HIF-1α were subcloned into the pCMV-Myc vector. Wild-type human HIF-2α was subcloned into the pCMV-Myc vector and the pCMV-HA vector. Human HIF-1α domains were subcloned into the pCMV-Myc vector.
9
Cloning and Characterization of Camelid MAVS
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Expression plasmids of Myc-tagged caMAVS and caRIG-I were constructed by inserting the full-length open reading frames (ORFs) into the pCMV-MYC vector with MYC fused to the N-terminus (Clontech, Takara Bio, Kusatsu, Shiga, Japan). HA and 3*Flag-tagged caMAVS constructs were cloned with Infusion technology (Vazyme Biotech Co., Ltd., China) into pCMV-HA (N-Terminal) (Clontech, Japan) and p3*FLag-CMV-10 (Sigma-Aldrich, USA) vectors, respectively. Truncated forms of caMAVS, which lacked the CARD domain (residues 10–77), PRR domain (residues 78–199), Transmembrane (TM) domain (residues 495–517), or Non-Characterized (NC) domain (residues 201–494) were constructed by using Infusion technology or overlapping extension PCR, as described previously [21 (link)]. Flag-tagged human MAVS (huMAVS) plasmid was used as a control and was kindly provided by Dr. Yuzhi Fu from the Wuhan Institute of Virology, Chinese Academy of Sciences. The PRDIII/I-luc and PRDII-Luc plasmids were purchased from Beyotime. pRL-TK (Promega Company, USA) expressing Renilla luciferase was used as endogenous transfection control to allow normalization.
10
Cloning and Expression of Sun4 Protein
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Total RNA was isolated from testicular suspensions of adult mice using TriFast™ (Peqlab Biotechnology) according to manufacturer's protocol. Reverse transcription was performed on 1 µg of total RNA with oligo(dT) primer and M-MLV reverse transcriptase (Promega). Based on the NCBI reference sequence NM_139151, a 5′-oligonucleotide upstream of the putative start codon (Sun4_5′) and a 3′-oligonucleotide downstream of the predicted stop codon (Sun4_3′) were used for PCR amplification of the full-length cDNA (Table S1 ). The product was cloned into the StrataClone Blunt PCR Cloning vector (pSC-B-amp/kan; Agilent Technologies) and verified by standard sequencing. For antibody production, a Sun4 fragment coding for amino acids 11-127 was amplified with primers S4_Nterm_5′_Nde and S4_Nterm_3′_Sal (Table S1 ) and cloned into pET-21a(+) expression vector (Novagen). To generate GFP- and Myc-tagged Sun4 constructs, the coding region corresponding to amino acids 135-444 was amplified with primers Sun4_ΔN_5′ and Sun4_SUNdom_3′ (Table S1 ) and cloned into either the pEGFP-C1 or the pCMV-Myc vector (Clontech Laboratories). Other constructs used in this study were described previously (Göb et al., 2010 (link)).
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