p21-Luc, pCMV-Bam-MDM2, and His-Ubiquitin (wt) have been described previously. myc-AIP4 was obtained from Dr. Tony Pawson (University of Toronto), and His-tagged ubiquitin (Ub) was provided by Dr. Wei Gu (Columbia University, USA). Ago2 was cloned into p3×Flag- CMV-10 (Sigma). miRNA expression vectors were constructed using the pCMV-MIR vector (Origene). All PCR products were confirmed by sequencing. Anti-p63 (DeltaNp63 (E6Q3O), Cell Signaling; Poly 938102 (anti-TAp63), BioLegend), anti-Ago2 (C34C6, Cell Signaling Technology), anti-Flag (M2, M3165, and M5, M4042, Sigma), anti-MDM2 (2A10, OP115, Calbiochem; SMP14, sc-965, Santa Cruz Biotechnology), anti-ubiquitin (550944, BD Bioscience), anti-Itch (ab109018, Abcam), anti-HA (sc-57592, Santa Cruz Biotechnology), anti-GFP (sc-390394, Santa Cruz Biotechnology), anti-p53 (Pab1801, sc-98, DO1, sc-126, Santa Cruz Biotechnology), and anti-actin (612657, BD Bioscience) were used according to the manufacturers’ instructions.
P3 flag cmv 10
Manufactured by Merck Group
Sourced in Japan
The P3×Flag-CMV-10 is a laboratory equipment product. It is a plasmid designed for the expression of proteins in mammalian cell lines. The plasmid contains a CMV promoter for strong, constitutive expression of the target protein, as well as a 3xFlag tag sequence for detection and purification purposes.
Automatically generated - may contain errors
Lab products found in correlation
Pgex 6p 1, by GE Healthcare (2 mentions)
Ab109018, by Abcam (1 mentions)
Pcmv mir vector, by OriGene (1 mentions)
Sc 390394, by Santa Cruz Biotechnology (1 mentions)
Sc 57592, by Santa Cruz Biotechnology (1 mentions)
Pegfp n3 expression vector, by Takara Bio (1 mentions)
Pcmv myc vector, by Takara Bio (1 mentions)
Infusion technology, by Vazyme (1 mentions)
Prl tk, by Promega (1 mentions)
Pfugw, by Addgene (1 mentions)
Pcmv myc, by Takara Bio (1 mentions)
Pmrx ip gfp lc3 rfp, by Addgene (1 mentions)
Pegfp lc3, by Addgene (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Pcdna3 ha, by Thermo Fisher Scientific (1 mentions)
Plexa, by Takara Bio (1 mentions)
Pb42ad, by Takara Bio (1 mentions)
Mcs bioid2 ha, by Addgene (1 mentions)
Quickmutation site directed mutagenesis kit, by Beyotime (1 mentions)
Lipofectamine ltx reagent, by Thermo Fisher Scientific (1 mentions)
10 protocols using p3 flag cmv 10
1
Ubiquitin-mediated p63 regulation
2
Plasmid construction for EGFP- and FLAG-tagged SOD1
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The construction of EGFP- and FLAG-tagged Cu/Zn superoxide dismutase 1 WT or G93A mutant plasmids was reported previously[55 (link)].
Amplified Cu/Zn superoxide dismutase 1 WT or G93A fragments were cloned into EcoRI and BamHI-digested p3×FLAG-CMV10 (Sigma, St. Louis, MO, USA) and the pEGFP-N3 expression vector (Clontech, Mountain View, CA, USA). EGFP-tagged beclin 1 plasmid was kindly provided by Dr. Qinjun Wang (University of Kentucky). A beclin 1-encoding cDNA was inserted into the EcoRI and BamHI sites of the pEGFP-N3 expression vector (Clontech). LC3 was amplified by PCR and was cloned into EcoRI-BamHI double-digested pEGFP-C1 (Clontech)[56 (link)].
Amplified Cu/Zn superoxide dismutase 1 WT or G93A fragments were cloned into EcoRI and BamHI-digested p3×FLAG-CMV10 (Sigma, St. Louis, MO, USA) and the pEGFP-N3 expression vector (Clontech, Mountain View, CA, USA). EGFP-tagged beclin 1 plasmid was kindly provided by Dr. Qinjun Wang (University of Kentucky). A beclin 1-encoding cDNA was inserted into the EcoRI and BamHI sites of the pEGFP-N3 expression vector (Clontech). LC3 was amplified by PCR and was cloned into EcoRI-BamHI double-digested pEGFP-C1 (Clontech)[56 (link)].
3
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.
4
Cloning and Characterization of pRNF114 and NS4B Constructs
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The pRNF114 gene was cloned into the p3×Flag-CMV-10 (Sigma-Aldrich), pFUGW (Addgene), pGEX-6P-1 (GE Healthcare), and pCMV-Myc (Clontech) vectors to generate pFlag-pRNF114, pFUGW-pRNF114, pGST-pRNF114, and pMyc-pRNF114, respectively. To generate site mutants, alanine was substituted for cysteine by PCR. The N- and C-terminal truncation mutants pRNF114(1-68) and pRNF114(69-228) were generated by standard PCR. The NS4B, NS4B(1-179), and NS4B(180-347) genes were cloned into the pCMV-Myc vector (Clontech) to obtain pMyc-NS4B, pMyc-NS4B(1-179), and pMyc-NS4B(180-347), respectively. The primers for amplification of plasmids are listed in Table 1 .
5
Cloning and Transfection of Porcine NDRG1
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The coding sequence of porcine NDRG1 was amplified from porcine white adipose cDNA and cloned into the p3×FLAG-CMV-10 (Sigma, St. Louis, MO) or pmCherry-C1 plasmid. The primers are listed in Table 1 . The plasmids pEGFP-LC3 (catalogue number 24920) and pMRX-IP-GFP-LC3-RFP (catalogue number 84573) were purchased from Addgene (Cambridge, MA, USA). All plasmids were transfected with Lipofectamine 3000 (Invitrogen, Grand Island, NY), according to the manufacturer’s instructions.
6
Plasmid Generation and Cloning
7
Construction of pFLG-VP2 Recombinant Plasmid
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The VP2 DNA fragment was amplified by PCR from the pAAV2-RC (Stratagene, USA), the upstream primer was TCG ATA GAT CTg ata tcG GCT CCG GGA AAA AAG AGG , and the downstream primer was CGg gat ccT TAC AGA TTA CGA GTC A (the italic letters were the cloning restriction sites of EcoRV and BamHI). Using p3×FLAG-CMV-10 (Sigma Aldrich) as the backbone vector, the VP2 sequence was inserted in-frame. The resulting construct was named pFLG-VP2. The DH5α strain of E coli was used for transformation and plasmid amplification. The successful FLAG and VP2 fusion in the pFLG-VP2 plasmid were identified by restriction enzyme digestion as resolved by 2% agarose electrophoresis and subsequent DNA sequencing.
8
Cloning and Mutagenesis of HSPB5
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cDNA of HSPB5 (NCBI No. NM_001289782) were amplified using a KOD One PCR kit (Toyobo, Osaka, Japan) and subcloned into the BamH I site of the p3×FLAG CMV-10 (Sigma-Aldrich) or MCS-BioID2-HA [14 (link)] (Addgene plasmid 74224) using in-fusion systems (Takara Bio, Shiga, Japan). Mutant HSPB5 (S19A), HSPB5 (S45A), HSPB5 (S59A), HSPB5 (3A; S19A, S45A, and S59A), and HSPB5 (R120G) [12 (link)] were also prepared by PCR based on in-fusion cloning.
9
Cloning and mutagenesis of RecQL4 and RNF8 proteins
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pFlag-CMV4-RecQL4, Flag-RecQL4-NT, Flag-RecQL4-HD, Flag-RecQL4-CT, and pcDNA3.0-HA-RNF8 were described previously22 (link),43 (link). RNF8 and RecQL4 fragments were subcloned into pEGFP-c1B. mCherry-tagged RNF8 was subcloned into pFlag-CMV4. The coding DNA sequences of WRAP53β were amplified by PCR with cDNA library of human U2OS cells and subcloned into p3×Flag-CMV-10 (Sigma). The coding DNA sequences of RNF8 and WRAP53β were amplified by PCR and subcloned into pGEX-6P-1 (GE Healthcare). RecQL4 mutants were generated with QuickMutation™ Site-Directed Mutagenesis Kit (Beyotime) according to the manufacturer’s protocol. To generate shRNF8 adenovirus constructs, a 19-mer RNA shRNA (ACATGAAGCCGTTATGAAT, accession no. NM_003958.3) was recombined into pAdEasy-1 vectors and RNF8-knockdown recombinant adenovirus was generated in HEK293 cells22 (link).
10
Cloning and Expression of PCV3 Cap Gene
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Plasmids expressing the Cap gene were constructed by cloning from the PCV3 infectious clone into pCMV-HA (#631604; Clontech), pEGFP-C1 (#U55763; Clontech), and p3 × FLAG-CMV-10 (#E4401; Sigma-Aldrich) vectors. The NPM1, nucleolin, and fibrillarin genes from PK15 cells were amplified and recombined into the lentivirus vector pWPXL (#12257; Addgene) to express the fusion proteins NPM1-GFP, nucleolin-GFP, and fibrillarin-GFP. LipofectamineTM LTX Reagent (#A12621; Thermo Fisher Scientific) was used for plasmid transfection. The primers are displayed in Table 1 .
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