The ORF of CiMex3A was separately inserted into pcDNA3.1 (+) (Invitrogen, USA), pEGFP-C1 (Invitrogen, USA), and p3×FLAG-Myc-CMV™-24 (Invitrogen, USA) plasmids. The mutants Mex3A (1–282) and Mex3A (1–454) were inserted into pcDNA3.1 (+) (Invitrogen, USA) and pEGFP-C1 (Invitrogen, USA), respectively. The ORF of RIG-I (GQ478334.2) was separately constructed into p3×FLAG-Myc-CMV™-24 and pEGFP-C1. The ORFs of TRIF (KC333648.1), PRMT6 (MN781672.1), and TLR3 (DQ864497.1) were separately inserted into pEGFP-C1. CDS regions of grass carp RAB5 (MF598473.1) and RAB7 (MF598474.1) were separately constructed into pDsRed2-C1 (Sangon Biotech, China). PKZ-GFP (36 (link)), ZDHHC1-GFP (37 (link)), TRAF6-GFP (38 (link)), pDsRED2-ER, and pDsRED2-Mitoplasmids (Biofeng) were stored in our lab. All the primers for vector construction are listed in Table 1
Pegfp c1
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
PEGFP-C1 is a plasmid vector that contains the gene encoding the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The vector is designed for the expression and detection of proteins fused to the C-terminus of GFP in mammalian cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (11 mentions)
Pegfp c1, by Takara Bio (5 mentions)
Pcdna3, by Thermo Fisher Scientific (4 mentions)
Pcdna3.1 flag, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Pgex 4t 1, by GE Healthcare (2 mentions)
Epha2 shrna, by GenePharma (2 mentions)
154cf media, by Thermo Fisher Scientific (1 mentions)
In fusion cloning kit, by Takara Bio (1 mentions)
Pegfp c1 plasmid, by Takara Bio (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
Pegfp n1, by Thermo Fisher Scientific (1 mentions)
Tagbfp, by Thermo Fisher Scientific (1 mentions)
Pgex 5x 1 2, by GE Healthcare (1 mentions)
Gfp trap dynabeads, by Proteintech (1 mentions)
Lds buffer, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by GoldBio (1 mentions)
Gfp trap dynabeads, by Thermo Fisher Scientific (1 mentions)
Halt phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
33 protocols using pegfp c1
1
Plasmid Constructs for Protein Expression
2
Hepatic Cell Line Culture and HCV NS4B Mutants
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293T, HepG2, Huh7.5.1, and HeLa cells were maintained in Dulbecco’s modified Eagle medium (DMEM) with 10 % fetal bovine serum (FBS) and cultured at 37 °C in a 5 % CO2 incubator. The replicon Con-1 was kindly provided by Prof. Ralf Bartenschlager (University of Heidelberg, Germany). To construct NS4B PBM mutants, the last four amino acids of NS4B were deleted or site-direct-mutated as alanine (AAAA) with appropriate sets of primers. The full-length cDNA of NS4B gene was designed as NS4BWT, the NS4B with last four amino acids deleted as NS4BD, and the NS4B with last four amino acids mutated as NS4BM. These NS4B sequences were inserted into pEGFPC1 and pFLAG-CMV2 plasmids (Invitrogen, USA) to construct pEGFPC1-NS4B, pEGFPC1-NS4BD, and pEGFPC1-NS4BM and pFLAG-CMV2-NS4B, pFLAG-CMV2-NS4BD, and pFLAG-CMV2-NS4BM, respectively. The plasmid pcDNA/Flag-Scribble (kindly provided by Prof. Rice, Baylor College of Medicine, USA) was used as template to amplify all the fragments of Scribble and cloned into indicated mammalian expression vectors. All the constructs were verified by DNA sequencing. The plasmids were transfected into cells at approximately 80 % confluence by Lipofectamine 2000 (Invitrogen, Karlsruhe, USA) in 150-μl DMEM medium without 10 % FBS, according to manufacturers’ instructions.
3
Generation and Characterization of Sufu Variants
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Sequence verified SUFU variants were created using the InFusion cloning kit (Clontech) and cloned into peGFP-C1 (Clontech) and pCDH (SBI) vectors. Sufu-null MEFs [2 (link)] and HEK-293T cells were cultured in DMEM media supplemented with 10% fetal bovine serum. ASZ001 [9 (link)] were cultured in 154CF media (Life Technologies) supplemented with 2% chelated fetal bovine serum and 0.05mM CaCl2.
For qRT-PCR experiments, each peGFP-C1-SUFU construct was nucleofected into Sufu-null MEFs using the Amaxa nucleofection kit and protocol, grown to confluency in DMEM + 10% fetal bovine serum, and then withdrawn from serum in DMEM for 24 hours to ensure each cell was in the G0 phase of the cell cycle. Nucleofection was done with 1 μg of DNA into 100 uL of media containing Sufu-null MEF cells and nucleofection reagent. After nucleofection, cells were plated onto a 24-well plate with 1 mL of DMEM supplemented with 10% fetal bovine serum per well. Cells were incubated for 24 hours then RNA was purified for qRT-PCR.
For qRT-PCR experiments, each peGFP-C1-SUFU construct was nucleofected into Sufu-null MEFs using the Amaxa nucleofection kit and protocol, grown to confluency in DMEM + 10% fetal bovine serum, and then withdrawn from serum in DMEM for 24 hours to ensure each cell was in the G0 phase of the cell cycle. Nucleofection was done with 1 μg of DNA into 100 uL of media containing Sufu-null MEF cells and nucleofection reagent. After nucleofection, cells were plated onto a 24-well plate with 1 mL of DMEM supplemented with 10% fetal bovine serum per well. Cells were incubated for 24 hours then RNA was purified for qRT-PCR.
4
Transient Gene Expression using Linear DNA
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Linear DNA (4-kbp) with the EGFP coding region was synthesized by PCR using the pEGFP-C1 plasmid (Clontech, Palo Alto, CA, USA) as a template. The following sequences of primer sets (Sigma-Aldrich Japan, Hokkaido, Japan) used were:
Forward primer: 5′-CCG CTA TCA GGA CAT AGC GT-3′
Reverse primer: 5′-TTG TCT GTT GTG CCC AGT CA-3′
Cells in 6-well plates were transfected using Lipofectamine2000 (Life Technologies) with 1 μg of pEGFP-C1 plasmid or linear 4-kbp DNA per well according to the manufacturer’s instructions. The transfected cells were trypsinized and collected 24 h after transfection. The EGFP gene expressions of each cell (at least 10,000 cells) were quantified by MACSquant Analyzer (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).
Forward primer: 5′-CCG CTA TCA GGA CAT AGC GT-3′
Reverse primer: 5′-TTG TCT GTT GTG CCC AGT CA-3′
Cells in 6-well plates were transfected using Lipofectamine2000 (Life Technologies) with 1 μg of pEGFP-C1 plasmid or linear 4-kbp DNA per well according to the manufacturer’s instructions. The transfected cells were trypsinized and collected 24 h after transfection. The EGFP gene expressions of each cell (at least 10,000 cells) were quantified by MACSquant Analyzer (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).
5
Plasmid Construction and Characterization
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The plasmid pOTB-UBIAD1 was purchased from Open Biosystems Inc. Enhanced green fluorescent protein (EGFP) vectors pEGFP-N1, pEGFP-C1, pCasper3-BG (TAGBFP-GFP), mammalian expression vector pcDNA3.1 and TAGBFP were obtained from Invitrogen. pDsRed-Golgi vector was obtained from Clontech, previously described28 (link). For construction of DsRed-H-Ras, full-length human H-Ras cDNA was amplified by PCR and cloned with DsRed-Monomer into the pcDNA3.1 vector. BFP-H-Ras was constructed by fusing H-Ras at the N-terminus of BFP into the pcDNA3.1 vector. GFP-H-Ras and its mutation were cloned into pEGFP-C1. Flag-H-Ras and its mutation were constructed by fusing the Flag tag to the N-terminus of H-Ras and inserting into pcDNA3.1. The plasmid 3Flag-UBIAD1 was constructed by fusing the 3Flag tag to the N-terminus of UBIAD1 and inserting into pcDNA3.1. pGFP-RBD was kindly provided by Dr. Mark R. Philips. Full-length human UBIAD1 and Apo E cDNA fragment were separately cloned into the pEGFP-N1 vector to create UBIAD1-EGFP and Apo E-EGFP. UBIAD1 was cloned into pcDNA3.1 to generate pcDNA3.1-UBIAD1. UBIAD1-BFP, Golgi-BFP and Apo E-BFP were constructed by fusing BFP with the C-terminus of UBIAD1 and Apo E and inserting into pcDNA3.1. All vectors for the BIFC assay were constructed using pcDNA3.1.
6
Plasmid construction and mutagenesis
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GFP-, DsRed-, Flag-, and GST-tagged
PAK4 were constructed by polymerase chain reaction (PCR) and sub-cloned into pEGFP-C1, DsRed, pcDNA3.1-Flag (Invitrogen), and pGEX-5X-1/2 (GE Healthcare, Bethesda, USA) vectors, respectively. The full length CORO1C was amplified by PCR using cDNA derived from SGC-7901 cells as a template and cloned into pEGFP-C1, DsRed, pcDNA3.1-Flag and pGEX-4T-2 vectors. CORO1C deletion constructs were obtained by PCR and cloned into pGEX-4T-2. The site-directed mutagenesis (PAK4S99A or PAK4S99D) was generated from PAK4 WT using the QuikChange kit (Stratagene, La Jolla, USA) according to the manufacturer′s instructions. eYFP-RCC2 was a kind gift from Prof. Sheng Xiao of Harvard Medical School (Boston, USA).
PAK4 were constructed by polymerase chain reaction (PCR) and sub-cloned into pEGFP-C1, DsRed, pcDNA3.1-Flag (Invitrogen), and pGEX-5X-1/2 (GE Healthcare, Bethesda, USA) vectors, respectively. The full length CORO1C was amplified by PCR using cDNA derived from SGC-7901 cells as a template and cloned into pEGFP-C1, DsRed, pcDNA3.1-Flag and pGEX-4T-2 vectors. CORO1C deletion constructs were obtained by PCR and cloned into pGEX-4T-2. The site-directed mutagenesis (PAK4S99A or PAK4S99D) was generated from PAK4 WT using the QuikChange kit (Stratagene, La Jolla, USA) according to the manufacturer′s instructions. eYFP-RCC2 was a kind gift from Prof. Sheng Xiao of Harvard Medical School (Boston, USA).
7
Purification and Analysis of GFP-RAD51AP1 Complexes
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The peGFP-RAD51AP1 expression vector is based on peGFP-C1 (Clontech) and has been described previously (Modesti et al., 2007 (link)). RAD51AP1 single or RAD54L/RAD51AP1 double KO cells were transfected with peGFP-C1 or peGFP-RAD51AP1 and Lipofectamine2000 (Invitrogen). Twenty-four hours after transfection, cells were subjected to a medium change, treated with 0.5 µM MMC for 2 h or 10 µM olaparib for 24 h. Cells were washed twice with warm PBS, fresh medium was added, and cells were incubated for the times indicated. Cells were lysed in chilled lysis buffer containing 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 0.5% NP-40, supplemented with EDTA-free protease inhibitor cocktail (Roche) and HALT phosphatase inhibitors (Thermo Fisher Scientific). For 1.5 × 106 cells, 25 μl of GFP-Trap® dynabeads (ChromoTek) were used to trap the ectopic proteins. Protein lysates were diluted to 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, and 0.1 unit DNase I (Gold Biotechnology) per µg protein, and mixed with the equilibrated beads at 4°C for 1 h with gentle rotation. The GFP-Trap® dynabeads were washed three times with 500 µl binding buffer, bound protein complexes were eluted in 40 µl 2× LDS buffer (Thermo Fisher Scientific) and fractionated on 7% NuPAGE Tris-Acetate gels (Thermo Fisher Scientific) and for Western blot analysis.
8
Plasmid Construction for Autophagy Research
9
Plasmid Construction for RBM4 and CAD Variants
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The plasmids pcDNA-FLAG-RBM4(a) and pEGFP-CAD (encoding residues 196–364 of RBM4a) have been described (2 (link)). Using a polymerase chain reaction (PCR)-based gene deletion strategy, Ala-tract #1 (residues 233–239) and #2 (residues 282–299) of pcDNA-FLAG-RBM4(a) were removed to generate pFLAG-RBM4-A0. The pFLAG-RBM4-A25 plasmid was constructed by substituting three nonalanine residues of Ala-tract #2 with alanines and inserting an additional seven alanines. The RBM4b-coding region was cloned into pcDNA3.1 in frame with the FLAG sequence. The coding regions of CAD-A0, CAD-A25, PSF and SRSF1/ASF were each inserted into vector pEGFP-C1 (Invitrogen) to generate the respective C-terminal green fluorescent protein (GFP) fusions. The pcDNA-FLAG-CoAZ was a kind gift of Lan Ko (Medical College of Georgia). The red fluorescent protein (RFP)-coding sequence was inserted into pcDNA-FLAG-RBM4(a) and pcDNA-FLAG-CoAZ to generate vectors RFP-RBM4(a) and RFP-CoAZ, respectively. The splicing reporter plasmids have been described (3 (link)).
10
Construction of pEGFPC1-NS4B Plasmid
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The pEGFPC1 was purchased from Invitrogen (Invitrogen, Carlsbad, CA, USA). Full length of NS4B gene with HCV genotype 1b was generated from Con1 plasmid which was kindly provided by Prof. Ralf Bartenschlager (University of Heidelberg, Germany) by using PCR. Then full length of NS4B was inserted into pEGFPC1 plasmid to construct pEGFPC1-NS4B plasmid.
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