The 5′ flanking region of the SELENBP1 promoter (−1584 to −34) was amplified by PCR from human genomic DNA. The PCR product was cloned into pGL2-Basic vector (Promega, Madison, WI, U.S.A.) to construct pGL2-SELENBP1/1584. The 1584-Lck was constructed by inserting both 5′-flanking regions of SELENBP1 promoter from pGL2-SELENBP1/1584 and Lck (lymphocyte-specific protein tyrosine kinase) cDNA into pHYK [50 (link)]. Deletion constructs containing different lengths of the 5′-flanking sequences of the SELENBP1 gene were generated by PCR amplification from the 1584-Lck plasmid. To express HBx transiently, the HA-tagged HBx gene was cloned into pRcCMV (Invitrogen, Waltham, NY, U.S.A.) to give pRcCMV-HBx.
Prc cmv
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PRc/CMV is a laboratory equipment product from Thermo Fisher Scientific. It serves as a core function in laboratory settings, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Glass coverslip, by Thermo Fisher Scientific (2 mentions)
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Pgl2 basic vector, by Promega (1 mentions)
Pgem3, by Promega (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Transit lt1 reagent, by Mirus Bio (1 mentions)
Lipofectamine reagent, by Thermo Fisher Scientific (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Timp 1, by R&D Systems (1 mentions)
Anti sirt1, by Merck Group (1 mentions)
Anti acetylated lysine, by Cell Signaling Technology (1 mentions)
Anti pcaf h 369, by Santa Cruz Biotechnology (1 mentions)
Anti gfp, by Abcam (1 mentions)
Anti histone h3 p ser10, by Merck Group (1 mentions)
Pflag cmv 2, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
17 protocols using prc cmv
1
SELENBP1 Promoter Characterization
2
Investigating TK1 Promoter Activity and Fhit Expression
3
Cloning and Construction of Gsc Mutant Constructs
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K197E17 (link) was obtained from Dan Kessler, Wnt11 constructs were from Kristen Kwan, Vangl2 from Ray Keller, and Prickle1 from Naoto Ueno. Gsc-GR has been described in ref. 89 (link). Fgf8, Fz7, Dvl2-GFP, dnRhoA and caRhoA constructs were provided by the Steinbeißer laboratory.
The following PCR primers were used for cloning of deletion constructs ∆HD and ∆GEH: ∆HDfor 5′-ATATCGATGCGCTGCAAGGAGTCGCTGCTG-3′, ∆HDrev 5′-CTGGACTCTGACAGTGGTCCTCGAGAT-3′, ∆GEHfor 5′-ATATCGATGCGCTGCAAGGAGTCGCTGCTG-3′, ∆GEHrev 5′-CTGGACTCTGACAGTGGTCCTCGAGAT-3′. The starting construct to clone T-Gsc was PML129 (vector backbone PGEM3, Promega), which contained the 658 bp Brachyury streak promoter, followed by a floxed LacZ cassette with triplicate polyadenylation signals to ensure that the downstream open reading frame is not part of the mRNA. To create construct T-Gsc the 771 bp Gsc coding sequence was inserted downstream, flanked by a 231 bp polyadenylation signal from the bovine growth hormone gene (from pRc/CMV, Invitrogen). Construct mT-Gsc was generated by mutating the Brachyury streak promoter 35 bp downstream of the transcriptional start site from TAAT into ACTG11 .
The following PCR primers were used for cloning of deletion constructs ∆HD and ∆GEH: ∆HDfor 5′-ATATCGATGCGCTGCAAGGAGTCGCTGCTG-3′, ∆HDrev 5′-CTGGACTCTGACAGTGGTCCTCGAGAT-3′, ∆GEHfor 5′-ATATCGATGCGCTGCAAGGAGTCGCTGCTG-3′, ∆GEHrev 5′-CTGGACTCTGACAGTGGTCCTCGAGAT-3′. The starting construct to clone T-Gsc was PML129 (vector backbone PGEM3, Promega), which contained the 658 bp Brachyury streak promoter, followed by a floxed LacZ cassette with triplicate polyadenylation signals to ensure that the downstream open reading frame is not part of the mRNA. To create construct T-Gsc the 771 bp Gsc coding sequence was inserted downstream, flanked by a 231 bp polyadenylation signal from the bovine growth hormone gene (from pRc/CMV, Invitrogen). Construct mT-Gsc was generated by mutating the Brachyury streak promoter 35 bp downstream of the transcriptional start site from TAAT into ACTG11 .
4
Recombinant hS360A-PC Purification and Characterization
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Recombinant hS360A-PC was obtained by PCR-based site-directed mutagenesis of hwt-PC cDNA. The cDNA of hwt-PC and hS360A-PC were inserted into the eukaryotic expression vector pRc/CMV (Invitrogen), transfected into HEK293 cells (CRL-1573 ATCC) and recombinant h(S360A)-PC was purified and characterized as described earlier [30] (link), [31] . The purity and integrity of h(S360A)-PC was evaluated by SDS-PAGE (Figure S1 ). h(S360A)-PC concentrations were quantified by measurement of absorbance at 280 nm using an absorption coefficient of 14.5 (280 nm, 1%, 1cm) and by ELISA, employing the horse anti-human PC polyclonal antibody PAHPC-H (HTI), as a catcher antibody and the horseradish peroxidase-conjugated anti-human PC polyclonal Dako P0374 as a detecting antibody [18] .
5
Stable Cell Line Generation for HBV
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Huh7 and HepG2 cells were transfected with pRc/CMV (Invitrogen, Carlsbad, CA), pCMV-pol or pCMV-HBV using TransIT-LT1 reagent (Mirus, Madison, WI) to generate Huh7-neo, Huh7-pol, Huh7-HBV, HepG2-neo, HepG2-pol and HepG2-HBV cell lines. The culture medium of transformants was collected for HBsAg measurement to confirm the success of stable transfection.
6
Establishment of Kv3.1 Expression in CHO Cells
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The method for establishing Kv3.1 expression in CHO cells has been previously described in detail [5 (link),22 (link)]. Briefly, rat Kv3.1b cDNA [23 (link)] was transferred into the expression vector pRc/CMV (Invitrogen Corporation, San Diego, CA, USA). CHO cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen Corporation) supplemented with 10% fetal bovine serum, 0.1 mM hypoxanthine, and 0.01 mM thymidine. Lipofectamine reagent (Invitrogen Corporation) was used to implant the Kv3.1b expression vector into CHO cells. Transfected cells were exposed to 500 µg/ml G418 (A.G. Scientific, San Diego, CA, USA), and antibiotic-resistant cells were selected and maintained in fresh IMDM containing G418. The cultures, incubated in 95% humidified air-5% CO2 at 37°C, were passaged every 4–5 days with a brief trypsin-EDTA (Invitrogen Corporation) treatment followed by seeding onto glass coverslips (diameter: 12 mm; Fisher Scientific, Pittsburgh, PA, USA) in a Petri dish. The cells were used for electrophysiology 12–24 h after the seeding. For electrophysiological recording, cell-attached coverslips were transferred to a continually perfused (1 ml/min) recording chamber (RC-13; Warner Instrument Corporation, Hamden, CT, USA).
7
MGL/CD301 Expression in K562 Cells
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The coding sequence of MGL/CD301 was inserted into a mammalian cell expression vector, pRc/CMV (Invitrogen), in both sense and anti-sense orientations. K562 cells were transfected with the plasmids by electroporation. After selection with Geneticin (G418 sulfate; Gibco), MGL/CD301-positive cells were enriched with immunomagnetic beads by using an anti-MGL/CD301 monoclonal antibody (mAb) MLD-1. Cloning of transfectants was performed by the limiting dilution method. Flow cytometric analysis with mAb MLD-1 (1:400-diluted ascites fluid) and FITC-labeled anti-mouse immunoglobulin G (Zymed, 5 μg/mL) was performed to determine the levels of MGL/CD301 expression on the obtained cell clones.
8
Chimeric Plasmids Encoding α-Catenin Mutants
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The plasmids (all in pRcCMV; Invitrogen) encoding chimeric protein EcΔ-Dn-α(280–906) presented in Fig. 2 were based on EcΔDn (Hong et al., 2010 (link)). The general maps of the EcΔ-Dn-α(280–906) chimera, Dn-α-catenin, Dn-αABD, and their mutants (also in pRcCMV) are presented in Figs. 2 , 3 , 4 , and 5 . The α-catenin mutation inactivating its vinculin binding site was constructed based on the structural studies (Choi et al., 2012 (link); Rangarajan and Izard, 2012 (link)). It includes five amino acid substitutions to alanine: R329A, R330A, L347A, L348A, and Y351A. The shRNA-target region of α-catenin (5′-CCTGTTCCATCTCAAATAA-3′) in the plasmids used in α-catenin–silenced A431 cells was modified using PCR-directed mutagenesis. The original plasmid encoding human αE-catenin was published (Troyanovsky et al., 2011 (link)). All plasmid inserts were verified by sequencing.
9
Construct Optimization via Mutagenesis
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10
Production and Purification of Recombinant Human CLU
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Recombinant forms of human CLU, LCN1 and TIMP1 were purchased from R&D Systems (Minneapolis, MN, USA). Natural CLU was purified by immunoaffinity chromatography of plasma prepared from human blood, as described [52 (link)]. A novel recombinant form of human CLU was developed starting from an expression construct previously described [48 (link)]. The novel expressed protein, rhCLU-αC-H2S, incorporates a hexahistidine and twin-strep tags at the C-terminus of the CLU α-chain, a modification to the original twin-strep tagged construct [48 (link)]. The full-length cDNA, carried in the plasmid pRc CMV (Invitrogen), was transiently expressed in human cells (MEXi-293E, IBI Lifesciences, Gottingen, Germany) and the secreted protein was purified from the cell culture medium according to previously published methods [48 (link)].
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