Human embryonic kidney 293 cell-derived 293A cells were purchased from Life Technologies (Carlsbad, CA, USA) and cultured in DMEM (Nacalai Tesque) supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France), 1% penicillin/streptomycin, 2 mM L -glutamine (Nacalai Tesque), and 1 × Non-Essential Amino Acid (Life Technologies) at 37°C in an atmosphere of 5% CO2 as described previously (Toyoda et al., 2016a (link)). All experiments were carried out with 293A cells at passages 10–20. To express human ABCG2 (NM_004827) fused with Myc-tag at its N-terminus (Myc-ABCG2) and EGFP (control), we used Myc-ABCG2 and EGFP-expressing adenoviruses constructed in our previous study (Ito et al., 2015 (link)), respectively. To express the URAT1, open reading frame of URAT1 (NM_144585.3) was cloned into a pcDNA3.1(+) vector (Life Technologies) with a FLAG tag at its N-terminus. To express mouse Abcg2 and EGFP (control), open reading frames of mouse Abcg2 (NM_011920) and EGFP were inserted into a pcDNA3.3 vector (Life Technologies), respectively.
Pcdna3.3 vector
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
PcDNA3.3 vector is a plasmid DNA vector commonly used for gene expression in mammalian cells. It contains a mammalian cytomegalovirus (CMV) promoter for high-level expression and a neomycin resistance gene for selection of transfected cells.
Automatically generated - may contain errors
Lab products found in correlation
Pcdna3.1 vector, by Thermo Fisher Scientific (2 mentions)
Geneart, by Thermo Fisher Scientific (2 mentions)
293a cells, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Nacalai Tesque (1 mentions)
Non essential amino acid (neaa), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Nacalai Tesque (1 mentions)
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (1 mentions)
Chymotrypsin, by Worthington (1 mentions)
Ez link nhs peg4 biotin, by Thermo Fisher Scientific (1 mentions)
Pierce nhs activated agarose, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (1 mentions)
Thermolysin, by Merck Group (1 mentions)
Histrap excel column, by GE Healthcare (1 mentions)
Amicon ultra 15, by Merck Group (1 mentions)
1 10 phenanthroline, by Merck Group (1 mentions)
6 protocols using pcdna3.3 vector
1
Overexpression of ABCG2 and URAT1 in 293A Cells
2
Bax Mutant Protein Expression and Purification
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All cell culture and transfection methods have been previously described.2 (link), 3 (link), 21 (link), 22 (link) Codon optimized WT human Bax and engineered mutant human Bax with the BH3 domain swapped for the BH3 domain of Bcl-xl (Bax-BH3sw) were synthesized and subcloned into pcDNA3.3 vector with the addition of a FLAG tag by GeneArt (Life Technologies, Grand Island, NY, USA). Mutant human Bax protein with all lysines replaced with arginines (Bax ∅-Lys) was engineered by GenScript (Piscataway, NJ, USA) and subcloned into pcDNA 3.1(+) vector (Invitrogen, Grand Island, NY, USA).3 (link) WT GFP-Bax was a kind gift from Dr Richard Youle (NIH, NINDS, Bethesda, MD, USA). Mutations in WT GFP-Bax at lysine 21, lysine 64 or both lysines 21 and 64 replaced with arginines (K21R, K64R and K21R/K64R, respectively) were engineered using site-directed mutagenesis. Primers for K21R: Forward, 5′-gctctgagcagatcatgaggacaggggcccttttgc-3′ Reverse, 5′-gcaaaagggcccctgtcctcatgatctgctcagagc-3′. Primers for K64R: Forward, 5′-gcgagtgtctcaggcgcatcggggacgaactgg-3′ Reverse, 5′-cgtccccgatgcgcctgagacactcgctcagc-3′. The K21R/K64R double mutant was generated sequentially using the same primers.
3
Overexpression of Human MGAT2 and MGAT3
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The full-length coding sequences of human MGAT2 and human MGAT3 are identical to NCBI accession numbers NM_025098 and NM_001109436, respectively. The respective sequences were subcloned into the pcDNA3.3 vector (Life Technologies, Carlsbad, CA) to generate expression plasmids in mammalian cells. To prepare overexpressed membranes, the expression vector was transiently transfected into COS-7 cells. After culture for 2 days, cells were collected and homogenized in ice-cold 20 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA and 300 mM sucrose. Cell homogenates were centrifuged (2000 rpm, 10 min, 4°C), and the supernatant was recovered. Total membrane fractions were isolated by ultracentrifugation (45,000 rpm, 60 min, 4°C). Pellets were resuspended in the same buffer and stored at -80°C. The protein concentration was determined using the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL) according to the instruction manual.
4
Production and Characterization of Engineered IgG1-DNP Antibody
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The IgG1‐DNP antibody consists of the variable domains of mouse monoclonal antibody (mAb) G2a2 against the hapten DNP combined with the constant domains of human IgG1 and the kappa light chain 17 . A triple mutant variant of the IgG1‐DNP was produced containing three mutations (E345R, E430G and S440Y) in the Fc domain, which enhance the ability of the antibody to form hexamers both in solution and on the cell surface (designated IgG1‐DNP‐RGY) 13 , 14 . Gene constructs for heavy and light chains were ordered separately (Thermo Fisher Scientific GeneArt, Regensburg, Germany) and cloned into a pcDNA3.3 vector (Thermo Fisher Scientific). Antibodies were expressed by transient transfection of Expi293F™ cells with equimolar amounts of heavy and light chain plasmid, using the ExpiFectamine™ 293 transfection kit (Thermo Fisher Scientific), according to the manufacturer’s guidelines. Secreted antibodies were harvested from the supernatant 5 days post‐transfection, 0·2 µm filtered and purified on a column of protein A sepharose. Solution phase hexamerization of IgG1‐DNP‐RGY was verified using high‐pressure size exclusion chromatography (HP‐SEC) analysis, as previously described 14 .
5
Purification and Activation of Kallikrein Enzymes
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All substrates and proteins, except for KLK1, KLK4, and KLK8, were purchased from commercial suppliers (Supplementary Table 1 ). For phage panning, chymotrypsin (Worthington Biochemical Corporation) was immobilized on resin using Pierce NHS-activated agarose (Thermo Fisher Scientific). Expression vectors encoding KLK1 (UniProt; P06870), KLK4 (UniProt; Q9Y5K2), and KLK8 (UniProt; O60259) were constructed with His tags at their C-termini, by introducing sequences into the pcDNA3.3 vector (Thermo Fisher Scientific). Pro-KLK1, KLK4, and KLK8 were produced in FreeStyle 293-F cells (Thermo Fisher Scientific) and purified using His Trap excel columns (GE Healthcare). Each enzyme was activated using thermolysin (Sigma-Aldrich) in activation buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5), then their reactions were stopped by adding 10 mM 1,10-phenanthroline (Sigma-Aldrich). After activation, buffers were exchanged for PBS using Amicon-Ultra 15 (10,000 NMWL; EMD Millipore). Biotinylation of proteins was performed using a 4-fold molar excess of EZ-Link NHS-PEG4-Biotin (Thermo Fisher Scientific) in PBS, then biotinylated proteins were purified using Amicon-Ultra 15.
6
Gene Silencing of lincRNACox2 in Macrophages
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For gene silencing, siRNAs for lincRNACox2 were synthesized and purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The following siRNAs were used: Mock siRNA sense, 5′-UAAGGCUAUGAAGAGAUACUU-3′ and antisense 5′-GUAUCUCUUCAUAGCCUUAUU-3′; si-lincRNACox2 siRNA sense, 5′-GCCCUAAUAAGUGGGUUGUUU-3′ and antisense, 5′-ACAACCCACUUAUUAGGGCUU-3′; and lincRNACox2 sense, 5′-AGTATGGGATAACCAGCTGAGGT-3′ and antisense, 5′-GAATGCTGAGAGTGGGAGAAATAG-3′. Macrophages were transfected with siRNAs (final concentration, 20 nM) using Lipofectamine RNAiMax reagent (cat. no. 13778030; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol for 24 h. The lincRNACox2 expression vector was generated by reverse transcription-quantitative PCR (RT-qPCR) amplification of lincRNACox2 cDNA using RNA from macrophages and cloned into a pcDNA3.3 vector (Thermo Fisher Scientific, Inc.). The promoter of lincRNACox2 was amplified by PCR from human macrophage genomic DNA. Macrophages were transfected with each reporter construct for 24 h, followed by assessment of luciferase activity.
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