Primary cortical neurons (DIV 10) were transfected using calcium phosphate.53 (link) For overexpression of bmf, cells were transfected with a plasmid encoding murine bmf (generously provided by Professor A Villunger, Innsbruck Medical University, Innsbruck, Austria).30 (link) A plasmid with enhanced GFP (eGFP-N1; Clontech, Saint-Germain-en-Laye, France) was used to allow the identification of transfected neurons for cell death assays. Cells were used for experiments 48 h after transfection.
Egfp n1
Manufactured by Takara Bio
Sourced in United States, Japan
EGFP-N1 is a plasmid vector that encodes the enhanced green fluorescent protein (EGFP) under the control of a CMV promoter. EGFP-N1 can be used for the expression and detection of EGFP-fusion proteins in mammalian cells.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (5 mentions)
Viafect transfection reagent, by Promega (2 mentions)
4.74 renilla luciferase expression vector, by Promega (2 mentions)
Dual glo luciferase reporter assay system, by Promega (2 mentions)
Mirus transit 2020, by Mirus Bio (2 mentions)
Dulbecco s modified eagle s media, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Biotin sp conjugated goat anti human igg, by Jackson ImmunoResearch (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Trichostatin a tsa, by Bio-Techne (1 mentions)
Tubacin, by Bio-Techne (1 mentions)
Sucrose, by Merck Group (1 mentions)
Quickchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Fugene hd, by Promega (1 mentions)
Dual luciferase kit, by Promega (1 mentions)
Renilla luciferase, by Promega (1 mentions)
Gene pulser, by Bio-Rad (1 mentions)
Opti mem, by Nepa Gene (1 mentions)
Egfp c1, by Takara Bio (1 mentions)
23 protocols using egfp n1
1
Transfection of Primary Cortical Neurons
2
Cloning and Modification of Fluorescent Proteins
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EGFP-N1 (Clontech) vectors were modified to incorporate the TagRFP fluorophore and/or a P2A sequence. FLAG constructs were created in a pHAN vector modified to include a FLAG sequence at the 3’ terminus of the polylinker. ECTM domains of δ-Pcdhs and Pcdhb11 were then cloned into the appropriate vector.
3
Recombinase-Mediated Cassette Exchange System
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PhiC31, R4, TP901-1, Bxb1 integrase and FLPe expression vectors were synthesised de novo and were driven by the CAG promoter. These codon sequences were optimised as previously described by Yamaguchi et al. (2011 (link)). Plasmid vectors used in the recombination assay were PhiC31neo-EGFP, R4neo-EGFP, TP901-1neo-EGFP, Bxb1neo-EGFP and FRTneo-EGFP. These EGFP vectors were based on the inspB4ins2 vector containing two HS4 dimers derived from pJC5-4 (a gift from Dr. G. Felsenfeld). The CAG promoter, EGFP open reading frame (ORF), and SV40 poly-A were ligated into a multiple cloning site between the HS4 dimers on inspB4ins2 (Ins2CAG-EGFP). The CAG promoter was derived from pCX-EGFP (a gift from Dr. Okabe, Osaka University, Japan), and the EGFP ORF and SV40 poly-A were derived from EGFP-N1 (Clontech, USA). The PhiC31neo-EGFP, R4neo-EGFP, TP901-1neo-EGFP, Bxb1neo-EGFP, or FRTneo-EGFP vectors were constructed by ligation of the ins2CAG-EGFP vector with relevant inserts digested from the pNeo-PhiC31 attB, pNeo-R4 attB, pNeo-TP901-1 attB, pNeo-Bxb1 attB, or pNeo-FRT vectors, respectively. The insCAG-ELuc vector (a gift from Dr. Nakajima, AIST, Japan) was used as the plasmid carrying the Emerald Luc (ELuc; TOYOBO, Japan) from the Brazilian click beetle Pynearinus termitilluminans (Nakajima et al. 2010 (link)) driven by the CAG promoter.
4
Luciferase Reporter Assay in 50B11 Cells
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50B11 cells were seeded at 10,000 cells/well in 48-well plates in 250μl of complete media and grown to 60–80% confluence. Cells were then transfected with each reporter construct (450ng) and 50ng pGL4.74 Renilla luciferase expression vector (Promega; Madison, WI) using ViaFect Transfection Reagent (Promega; Madison. WI) in 25ul Opti-MEM (ThermoScientific, Waltham, MA) with a 4:1 ratio in 250μl complete medium. The transfection efficiency of 50B11 cells was evaluated by transfecting cells with EGFP-N1 (Clontech; Mountain View, CA) in parallel reactions. At 48 h post-transfection, Firefly and Renilla luciferase were measured using the Dual-Glo Luciferase reporter assay system (Promega). The amount of Firefly luciferase normalized to the Renilla luciferase and expressed as the relative fold difference of the empty pGL3 promoter vector. Each enhancer construct was tested in quadruplicate.
5
Detecting Anti-MOG Antibodies in Live Cells
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MOG-Abs were detected in a live cell assay, as described.11 (link),20 Briefly, HeLa cells were transiently transfected with human full-length MOG fused C-terminally to enhanced green fluorescent protein (EGFP)-N1 (Clontech Laboratories, Mountain View, CA) or with EGFP alone (control cells). As secondary reagents, biotin-SP-conjugated goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) and Alexa Fluor 647–conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA) were applied. For the determination of anti-MOG reactivity, we gated on cells with an FITC fluorescence intensity above 500 and determined their mean fluorescence intensity (MFI) in the allophycocyanin channel. For serum (diluted 1:50), we calculated the MFI ratio between MOG-EGFP–transfected cells and cells transfected with EGFP alone. For cell culture supernatants (used undiluted), the MOG reactivity was determined as delta MFI (reactivity to MOG-transfected cells—reactivity to control transfected cells) because the reactivity to control cells of the cell culture supernatant was close to zero. Negative delta MFI was considered as zero. Threshold was set to mean +3 SD of the values from controls. Values beyond mean +5 SDs were not included in the threshold calculation. The recognition of epitopes on MOG was determined with a panel of mutated variants of MOG essentially as described.21 (link)
6
Cloning and Expression of Spastin Constructs
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Mouse cDNA encoding full-length spastin (M1; clone MGC: 54786, clone sequence BC046286.1) was cloned in EGFP-N1 (Clontech, Mountain View, CA, USA) or pCMV-FLAG-MAT-Tag-2 expression vector (Sigma-Aldrich) or mCherry-N1 (a generous gift from Roger Tsien, University of California San Diego, San Diego, CA, USA). cDNA lacking the first 84 aa (M85), cDNA encoding M1 lacking the AAA ATPase cassette (aa 1-338; M1Δ) and cDNA encoding the first 84 aa (N-Ter) were amplified from the M1 construct by PCR using specific primers and cloned in EGFP-N1 or mCherry-N1. To generate point mutation c.445C>Y in M1 and M85 constructs, the QuickChange II XL site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) was used according to the manufacturer's instructions. The correct sequence of all constructs and the presence of the point mutation were verified by DNA sequencing. mRFP-VAMP7 was previously described (Burgo et al., 2009 (link); Tsaneva-Atanasova et al., 2009 (link)). Tubulin-m-Cherry was a generous gift from Roger Tsien.
Sucrose (Sigma-Aldrich), nocodazole (Sigma-Aldrich), tubacin (Tocris, Bristol, UK) or trichostatin A (TSA; Tocris) were added to culture medium at different final concentrations. When needed, drugs were kept as stock solution in dimethyl sulfoxide and working dilutions were prepared freshly on each day of the experiment.
Sucrose (Sigma-Aldrich), nocodazole (Sigma-Aldrich), tubacin (Tocris, Bristol, UK) or trichostatin A (TSA; Tocris) were added to culture medium at different final concentrations. When needed, drugs were kept as stock solution in dimethyl sulfoxide and working dilutions were prepared freshly on each day of the experiment.
7
Plasmid Construction and Transformation in E. coli
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Four different plasmids were used in this study, EGFP-N1, EGFP-pIRES, EGFP-pIRES-mCnB, and EGFP-pIRES-mCnA. EGFP-N1 is a commercial product from Clontech. mCnB and mCnA genes were amplified from total RNA as previously described (Wang et al., 2008 (link), Yoshiga et al., 2002 (link)), and cloned into the EGFP-pIRES plasmid with 5′ Nhe Ⅰ and 3′ Sac Ⅰ restriction sites, which were later used to confirm successful insertion.
Recombined plasmid DNAs were transfected into HB101 competent Escherichia coli. Individual colonies were transferred into Kanamycin (20 µg/ml) LB medium and incubated over night at 37 °C with vigorous shaking (250 rpm on a rotary shaker) until the bacteria reached late log phase. Plasmid DNAs were prepared using alkaline lysis with SDS were purified with polyethylene glycol (PEG, 40% PEG6000, 30 mM MgCl2) and were recovered with deionized distilled H2O or 1 × TE buffer (pH 7.6).
Recombined plasmid DNAs were transfected into HB101 competent Escherichia coli. Individual colonies were transferred into Kanamycin (20 µg/ml) LB medium and incubated over night at 37 °C with vigorous shaking (250 rpm on a rotary shaker) until the bacteria reached late log phase. Plasmid DNAs were prepared using alkaline lysis with SDS were purified with polyethylene glycol (PEG, 40% PEG6000, 30 mM MgCl2) and were recovered with deionized distilled H2O or 1 × TE buffer (pH 7.6).
8
Evaluating Transfection Efficiency in HEK293 Cells
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HEK293 cells were seeded at 20,000 cells/well in 48-well plates in 250 μl of complete media and grown to 60–70% confluence. Cells were then transfected with each reporter construct (450 ng) and 50 ng pGL4.74 Renilla luciferase expression vector (Promega; Madison, WI) using ViaFect Transfection Reagent (Promega; Madison. WI) in 25 μl Opti-MEM (ThermoScientific, Waltham, MA) with a 4:1 ratio in 250 μl complete medium. The transfection efficiency of HEK293 cells was evaluated by transfecting cells with EGFP-N1 (Clontech; Mountain View, CA) in parallel reactions. 48 h post transfection, Firefly and Renilla luciferase activities were measured using the Dual-Glo Luciferase reporter assay system (Promega). Firefly luciferase activities were normalized to the Renilla luciferase activity and expressed as the relative fold difference of the empty pGL3 promoter vector. Each luciferase construct was tested in quadruplicate.
9
Profiling FMRpolyG Protein Expression
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HEK293 cells were transfected with constructs expressing FMRpolyG-GFP, ATG-FMRpolyG-GFP, FMRpolyA, or EGFP-N1 (Clontech) using Fugene HD (Promega) following the manufacturer’s protocol [24 (link), 26 (link)]. Lysates were collected 24 h after transfection, as previously described [25 (link), 26 (link), 30 (link)]. Proteins were separated using SDS-PAGE and transferred to PVDF membrane. Proteins were detected with antibodies against GFP (Sigma, 1:1000) and FMR polyclonal antibodies (11000). GAPDH or tubulin was used as a loading control.
10
FOXL2 Fusion Protein Expression
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Since FOXL2 is a single‐exon gene, its full‐length open reading frame was amplified from the genomic DNA of patients using primers incorporating restriction enzyme sites (EcoRI‐KpnI). The amplified gDNA products were purified, digested, and then cloned into the digested plasmids of phosphorylated enhanced green fluorescent protein (EGFP)‐N1 (Clontech, CA, USA), leading to the production of fusion proteins with the EGFP on the C terminus of FOXL2 (the former codon of termination codon in the wild‐type FOXL2 or predicted termination codon in mutant FOXL2 followed by the open reading frame of EGFP, which were designated as FOXL2‐WT‐EGFP, FOXL2‐Pro156Argfs‐EGFP, and FOXL2‐ Ala330Glyfs‐EGFP, respectively). All expression constructs were sequenced to confirm the presence of the desired mutations and exclude PCR‐induced mutations.
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