Constructs expressing myc-tagged or EGFP-tagged full-length TRIM3 and ΔRBCC-TRIM3 (amino acids 283–744) were generated in pCDNA3.1-Myc (Invitrogen) and pEGFP-N1 (Clontech), respectively. The EGFP-PURA construct was a gift of J.L. Napoli (University of California, Berkeley, Berkeley, CA; Chen et al., 2008 (link)).
Pcdna3.1 myc
Manufactured by Thermo Fisher Scientific
Sourced in United States
PcDNA3.1‐Myc is a mammalian expression vector that allows for the expression of recombinant proteins in a variety of cell lines. The vector contains a cytomegalovirus (CMV) promoter for high-level expression and a c-Myc epitope tag for detection and purification of the expressed protein.
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Lab products found in correlation
Pegfp n1, by Takara Bio (1 mentions)
Pegfp c2, by Takara Bio (1 mentions)
Talon metal affinity resin, by BD (1 mentions)
Pcdna3.1 ha, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Ha ubiquitin, by Addgene (1 mentions)
Pcdna3.1 flag vector, by Thermo Fisher Scientific (1 mentions)
Pdsred monomer membrane, by Takara Bio (1 mentions)
Pdsred er, by Takara Bio (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Pcdna3, by Takara Bio (1 mentions)
Pegfp n1 vector, by Takara Bio (1 mentions)
Plasmid midi kit, by Qiagen (1 mentions)
Pef dest51 vector, by Thermo Fisher Scientific (1 mentions)
10 protocols using pcdna3.1 myc
1
Constructing Myc- and EGFP-tagged TRIM3
2
Importin-α1 and Importin-β Purification
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The cDNA of importin-α1 was cloned into pEGFP-C2 (Clontech), pET-28a (Novagen) and pcDNA3.1-Myc (Invitrogen) to generate GFP-, His-, and Myc-tagged importin-α1. The pEGFP-C2-importin-α1 and pcDNA3.1-Myc-importin-α1 plasmids were used as templates for site-directed mutagenesis to generate the mutations at T9A, S62A, T9E, S62E, 2A and 2D. The cDNA of importin-β was cloned into pGEX4T-1 (Novagen) to generate GST-tagged importin-β. pET28a-KIFC1 887-end was kindly provided by Shuli Zhang and pEGFP-NT was a kind gift from Qinying Liu (both at College of Life Sciences of Peking University, China). E. coli expressing His–importin-α1 and His–KIFC1 887-end were purified by TALON Metal Affinity Resin (BD Biosciences Clontech) according to the manufacturer's instructions. E. coli expressing GST–importin-β was purified using glutathione–Sepharose-4B (Pharmacia, Pfizer Inc., NY) according to the manufacturer's instructions. The proteins were desalted using an Amicon ultral-4 centrifugal filter (Millipore) and were resuspended in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4).
3
Generating WISP3 and β-catenin Constructs
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The coding sequence for the full length of WISP3 was cloned into the pcDNA3.1‐Myc plasmid (Invitrogen) to generate the WISP3 expression vector. Then, the WISP3 coding sequence and the myc tag were subcloned into pRDI‐292 lentiviral expression vector, and cells were selected with puromycin. The coding sequence for β‐catenin with substitute mutations of T41A and S45A was cloned into pcDNA3.1‐Myc. The resulting plasmid was transfected into cells by FuGENE6 (Roche, Basel, Switzerland), and the transfected cells were selected in the presence of G418. Resistant clones were further confirmed the expression of β‐catenin by Western blot. The coding sequence for luciferase was cloned into FG12 expressing vector. PVTT‐1 and QSG‐7701 cells were labelled with luciferase for metastasis assay.
4
Characterizing PHB2 and BI1 Domains
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PHB2 possesses 299 amino acids (aa), including a N-terminal mitochondrial targeting domain (N, 1-50 aa), a PHB domain (PHB, 68-185 aa), a coiled coil domain (CC, 190-264 aa), and a C-terminal region (C, 265-299 aa). BI1 contains 237 aa with a N-terminal domain (N, 1-29 aa), several transmembrane domains (TM, 30-222 aa) and a C-terminal domain (C, 223-237 aa). PHB2 and BI1 sequences were amplified from the cDNA of human HK2 cells. Amino acids 51-299 (PHB2ΔN), 1-264 (PHB2ΔC), 1-189 and 265-299aa (PHB2ΔCC), 1-67 and 186-299aa (PHB2ΔPHB) and full length (1-299 aa) of PHB2 were amplified using PCR and the produces were introduced into pcDNA3.1/Myc (Invitrogen) to construct the Myc-PHB2 mutants. Similarly, BI1ΔN (30-237 aa), BI1ΔC (1-222 aa), BI1ΔTM (1-29 and 223-237 aa) and full length (1-237 aa) of BI1 were inserted into pcDNA3.1/HA (Invitrogen) to generate BI1 mutants. siRNAs against BI1 (BI1-si), TIM23 (TIM23-si), and PHB2 (PHB2-si) were designed and synthesized by GenePharma Co, Ltd. (Shanghai, China). The control siRNA (Ctrl-si) was employed as a negative control under similar conditions. Transfection with plasmids or siRNA was performed using Lipofectamine 2000 (Invitrogen) based on our previous studies 31 (link), 32 (link).
5
Cloning and Mutagenesis of GSK3β, DISC1, and TRAX
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The pcDNA3.1-rTRAX-V5, pcDNA3.1-ΔNLS-rTRAX-V5 and pSuper-shTRAX-F constructs were developed as previously described [24 (link)]. The DNA fragments of rat GSK3β and human GSK3β were amplified from cDNA of PC12 cells and HEK293T cells, respectively, and cloned into pcDNA3.1-Myc (Invitrogen) using polymerase chain reaction (PCR) and the primers described in Table S1 . The DNA fragment of rat DISC1 was amplified from the cDNA of PC12 cells by PCR and cloned into pCMV-Tag2B (Stratagene, La Jolla, CA, USA) using the primers listed in Table S1 .
The point mutation of GSK3β-S9A was created by a method described earlier [36 (link)] using standard protocols and site-specific primers (5'-GACCGAGAACCACCG- CCTTTGCGGAGAGC-3' and 5'- GCTCTCCGCAAAGGCGGTGGTTCTCGGTC-3'). pLKO1-h-shTRAX and pLKO1-r-shC1D were purchased from the NRC, Academia Sinica (Taipei, Taiwan).
The point mutation of GSK3β-S9A was created by a method described earlier [36 (link)] using standard protocols and site-specific primers (5'-GACCGAGAACCACCG- CCTTTGCGGAGAGC-3' and 5'- GCTCTCCGCAAAGGCGGTGGTTCTCGGTC-3'). pLKO1-h-shTRAX and pLKO1-r-shC1D were purchased from the NRC, Academia Sinica (Taipei, Taiwan).
6
Cloning and Mutagenesis of Eag1 and MKRN1
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cDNAs for rat Eag1 (Dr Olaf Pongs, Saarland University, Germany) and MKRN1 long isoform were subcloned into pcDNA3.1, pcDNA3.1-Myc, or pcDNA3.1-Flag vectors (Invitrogen). Disease-causing (G348R, G469R, and I467V) and glycosylation-deficient Eag1 mutations (N388Q and N406Q; Eag1-QQ), as well as MKRN1 short form and the catalytically inactive E3 ligase mutant MKRN1-H307E, were generated using the QuikChange site-directed mutagenesis kit (Agilent Technologies), followed by verification with DNA sequencing. The other cDNA constructs include pDsRed-Monomer-Membrane (Clontech), pDsRed-ER (Clontech Laboratories), and HA-Ubiquitin (Addgene).
7
Cloning and Mutagenesis of Human TNNT3
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Total RNA from human skeletal muscle (previously collected from aborted fetuses affected with embryo arrest and stored at −80°C in our laboratory) and the reverse transcription reaction were used to generate cDNA by the PrimeScript™ RT reagent kit with gDNA Eraser. PCR amplification was conducted with the corresponding primers (listed in Table S1 ). The fragments containing the entire coding sequence of human TNNT3 were cloned into the appropriate sites (EcoRI/HindIII or HindIII/KpnI) of the pcDNA3.1‐Myc (Invitrogen, Carlsbad, CA, USA) and pEGFP‐N1 vector (Clontech, San Jose, CA, USA) to create pcDNA3.1/TNNT3‐Myc and pEGFP‐N1/TNNT3 (translational fusion with a green fluorescence protein encoding gene), respectively. The variant c.187C > T (p.R63C) was introduced to TNNT3 cDNA by Site‐Directed Mutagenesis Kit following the manufacturer's instructions. The pcDNA3.1/TNNT3‐Myc or pEGFP‐N1/TNNT3 was used as a template. After confirming the sequences, they were designated as pcDNA3.1/TNNT3, pcDNA3.1/TNNT3‐mut, pEGFP‐N1/TNNT3, and pEGFP‐N1/TNNT3‐mut. Plasmids were purified by using a Plasmid Midi Kit (Qiagen).
8
Protein Expression Constructs in E. coli and Mammalian Cells
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For recombinant protein expression (of all p63, p73, and DARPin constructs) in E. coli a pET15b vector was used (Novagen, Merck KGaA). PCR-generated inserts were introduced in pET-15b-His10-TEV, pET-15b-His10-TEV-Avi or pET-15b-His10-TEV-HA (N-terminal His10-tag followed by a tobacco etch virus (TEV) protease cleavage site (and Avi- or HA-tag)) by subcloning using BamHI and XhoI restriction sites. For transient expression in mammalian cells, PCR-generated inserts were introduced in pcDNA3.1(+) Myc (Invitrogen, Thermo Fisher Scientific Inc) by subcloning PCR-generated inserts using BamHI and XhoI.
For the cloning of the leucine zipper-DARPin constructs the protein-coding sequence, including the DARPin, fused at its C-terminus to a (G4S)4 linker, followed by the leucine zipper sequence (amino acids 250–281 of yeast GCN4) was synthesized (Eurofins). This construct was inserted into a pET-15b-His10-TEV vector using BamHI and XbaI.
For the cloning of the leucine zipper-DARPin constructs the protein-coding sequence, including the DARPin, fused at its C-terminus to a (G4S)4 linker, followed by the leucine zipper sequence (amino acids 250–281 of yeast GCN4) was synthesized (Eurofins). This construct was inserted into a pET-15b-His10-TEV vector using BamHI and XbaI.
9
SCOTIN Protein Expression and Purification
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The coding sequence of FL human SCOTIN was cloned into the pcDNA3.1‐Myc (Invitrogen, V80020) or pEF‐DEST51 vector (Invitrogen, 12285011) with a C‐terminal DsRed monomer (among pDsRed‐monomer‐N1; Clonetech, 632465) tag. SCOTIN mutants lacking the PRD or amino acids 150–177 were generated with a QuickChange site‐directed mutagenesis kit (Stratagene, 200519) according to the manufacturer's protocol. Str‐Ii‐VSVG‐SBP‐EGFP (Addgene, 65300; a gift from Franck Perez), Rab5Q79 L‐EGFP (Addgene, 28046; a gift from Qing Zhong), and Rab7‐EGFP (Addgene, 28047; a gift from Qing Zhong) were acquired from Addgene. VSVG in Str‐Ii‐VSVG‐SBP‐EGFP was replaced with SCOTIN or SCOTIN(Δ150‐177) to construct Str‐Ii‐SCOTIN‐SBP‐EGFP or Str‐Ii‐SCOTIN(Δ150‐177)‐SBP‐EGFP, respectively. Str‐Ii‐SCOTIN‐SBP‐DsRed and Str‐Ii‐SCOTIN(Δ150‐177)‐SBP‐DsRed were cloned by replacing EGFP with the DsRed monomer. All constructs were confirmed by DNA sequencing. The PRD and PRD(Δ150‐177) expression plasmids used for protein purification were generated using Gibson assembly with the DNA fragment amplified from the human SCOTIN cDNA containing a TEV cleavage site into the pET28a vector.
10
Constructing SCOTIN-fusion Proteins
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To construct mEmerald‐SCOTIN(PRD)‐Sec61β, the PRD of human SCOTIN was added at the BglII site of mEmerald‐Sec61β‐C1 (Addgene, 90992; a gift from Jennifer Lippincott‐Schwartz) using Gibson assembly. To construct pcDNA3.1‐CD63‐SCOTIN (PRD)‐Myc, the PRD of human SCOTIN and the human CD63 coding sequence were cloned into pcDNA3.1‐Myc (Invitrogen, V80020). pcDNA3.1‐CD63‐SCOTIN(PRD)‐YFP was cloned by replacing Myc with YFP in pcDNA3.1‐CD63‐SCOTIN(PRD)‐Myc. To construct MTS‐SCOTIN(PRD)‐YFP, CD63 in pcDNA3.1‐CD63‐SCOTIN(PRD)‐YFP was replaced with the coding sequence of human TOM20 (N‐terminal 33 amino acids).
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