The human ERα coding sequence was amplified from pcDNA3.1-human ERα66 [5 (link)] using KOD -Plus- Neo polymerase with restriction site-adapted primers, cloned into a pCMV-Tag 2B vector (Agilent Technologies, Santa Clara, CA, USA), and fused in-frame with a FLAG epitope tag sequence (pFLAG-hERα66). Human, mouse, and rat ERβ coding sequences were amplified from the pGEM-T-Easy vectors prepared above and cloned into pCMV-Tag 2B vectors (pFLAG-hERβ1, pFLAG-mERβ1, pFLAG-mERβins, pFLAG-rERβ1, and pFLAG-rERβins). The cloned fragments were confirmed by DNA sequencing analyses.
Pcmv tag2b vector
Manufactured by Agilent Technologies
Sourced in United States
The PCMV-Tag2B vector is a plasmid used for the expression and purification of recombinant proteins in mammalian cell lines. It contains a cytomegalovirus (CMV) promoter for high-level expression, as well as a C-terminal tag sequence for detection and purification of the expressed protein.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (6 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (3 mentions)
Dharmafect transfection reagent, by Horizon Discovery (2 mentions)
Pcmv ha vector, by Takara Bio (2 mentions)
Viafect transfection reagent, by Promega (2 mentions)
Pcmv myc vector, by Takara Bio (2 mentions)
Lamp1 mcherry plasmid, by Addgene (2 mentions)
X tremegene 9, by Roche (2 mentions)
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Pgex 4t 1, by GE Healthcare (1 mentions)
Ha ezh2, by Addgene (1 mentions)
Ha suz12, by Addgene (1 mentions)
Pentr1a vector, by Thermo Fisher Scientific (1 mentions)
Virapower adenoviral expression system, by Promega (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Dual luciferase reporter assay, by Promega (1 mentions)
8 protocols using pcmv tag2b vector
1
Cloning and Characterization of Estrogen Receptors
2
Plasmid Cloning and Expression of Histone Modifiers
3
Plasmid Construction and Transfection Protocols
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pCMV-DiZPK-PylRS plasmid encoding a DiZPK-aminoacyl-tRNA synthetase/tRNA pair was kindly provided by Professor Peng R. Chen at Peking University (Zhang et al., 2011 (link)) and is available on Addgene (plasmid #91706). Human IFITM3 cDNA was purchased from Open Biosystems and PCR cloned into pCMV-HA vector (TaKaRa) using EcoRI and SalI as the restriction sites. All mutants of IFITM3 were generated by QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, 200523) using primers listed in Table S2 . VCP cDNA was purchased from Open Biosystems and PCR cloned into pCMV-Tag2B vector (Agilent Technologies) using BamHI and HindIII as the restriction sites and pCMV-myc vector (TaKaRa) using SalI and KpnI as the restriction sites. LAMP1-mCherry plasmid (Van Engelenburg and Palmer, 2010 (link)) was obtained from Addgene (plasmid #45147).
ViaFect transfection reagent purchased from Promega or Lipofectamine 3000 from ThermoFisher Scientific was used for transfection of Hela cells, and Xtremegene 9 purchased from Roche was used for transfection of HEK293T cells. For RNA interference, siRNAs were synthesized by Genepharma and transfected into cells using DharmaFECT transfection reagent (Dharmacon).
siRNA sequences are as follows:
Control: CGTACGCGGAATACTTCGA;
VCP: AACAGCCAUUCUCAAACAGAATT (Kirchner et al., 2013 (link));
UBXD1: CCAGGUGAGAAAGGAACUUTT (Kirchner et al., 2013 (link)).
ViaFect transfection reagent purchased from Promega or Lipofectamine 3000 from ThermoFisher Scientific was used for transfection of Hela cells, and Xtremegene 9 purchased from Roche was used for transfection of HEK293T cells. For RNA interference, siRNAs were synthesized by Genepharma and transfected into cells using DharmaFECT transfection reagent (Dharmacon).
siRNA sequences are as follows:
Control: CGTACGCGGAATACTTCGA;
VCP: AACAGCCAUUCUCAAACAGAATT (Kirchner et al., 2013 (link));
UBXD1: CCAGGUGAGAAAGGAACUUTT (Kirchner et al., 2013 (link)).
4
Plasmid Constructs and RNA Interference for IFITM3 and VCP
5
EGFP-MAGI1 Construct Generation
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EGFP-MAGI1 construct (The GenBank gene accession number: KY651081 ) was a kind gift from Dr. Mochizuki (Department of Cell Biology, National Cardiovascular Center Research Institute, Osaka, Japan) (13 (link)). pCMV-FLAG-tagged-MAGI1-WT was generated by sub-cloning MAGI1 from EGFP-MAGI1 into the pCMV-Tag2B vector (Agilent Technologies, Santa Clara, CA, USA) at sites recognized by restriction enzymes EcoRI and HindIII. pCMV-FLAG-tagged-MAGI1-S741A mutant was generated by site-directed mutated pCMV- FLAG-tagged-MAGI1-WT using a QuikChange site-directed mutagenesis kit (Agilent Technologies) according to the manufacturer's instructions. FLAG-tagged adenoviral vectors containing MAGI1-WT and -S741A mutant were generated by cloning each corresponding insert from pCMV- FLAG-tagged-MAGI1-WT and -S741A into the pENTR1A vector (Life Technologies) at sites recognized by the restriction enzymes KpnI and NotI, and then a recombinase reaction was performed to get a pDEST-based vector following manufacture's instruction (#K4930-00, ViraPower Adenoviral Expression System, Promega). Where indicated, an adenovirus containing β-galactosidase (Ad-LacZ) was used as a control (42 (link)).
6
Cloning and Mutagenesis of UBAP1 in Neuro-2a Cells
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The coding region of wild-type UBAP1 (GenBank NM_016525.4) was amplified by PCR from human kidney first-strand cDNA. The wild-type UBAP1 was cloned into a pCMV-Tag2B vector (Agilent Technology) at the sites of BamHI and Hind III. The primers used in the UBAP1 construct were as follows: sense 5’-TGACGGATCC ATGGCTTCTAAGAAGTTGGG-3’ and antisense 5’-TGACAAGCTT TCAGCTGGCTCCTGCCCGAG-3’ . The A157* and L171* mutants were separately constructed by site-directed mutagenesis into the aforementioned vector. All constructs were confirmed by Sanger sequencing.
Neuro-2a cells were obtained from Stem Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2. Cellular transfections were performed with Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocols.
Neuro-2a cells were obtained from Stem Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2. Cellular transfections were performed with Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocols.
7
Nanog Promoter Activation Assay
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E14tg2a cells were plated in 24-well plates and were transfected with Nanog-5P, which contains 2.5 kb of the 5’ promoter region of the mouse Nanog gene (Addgene), pCMV-2B-Bcl3 using Lipofectamine 3000 reagent in accordance with the manufacturer’s protocol (ThermoFisher Scientific). pCMV-2B-Bcl3 was cloned into the multiple cloning site (BamHI and XhoI) of the pCMV Tag 2B vector (Agilent technologies). The cells were extracted 24 h after transfection and the extracted cells were analyzed by using the Dual-Luciferase Reporter Assay (Promega), with the luciferase activity measured in a multilabel plate reader (VICTOR3). The relative luciferase activity of each sample was normalized to Renilla activity.
8
Recombinant Expression Vector Construction
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The expression vectors for FLIP and Bcl-2 were amplified by polymerase chain reaction, digested with appropriate restriction enzymes, and cloned in-frame into a pCMV-Tag 2B vector (Agilent Technology). Then, the constructs were confirmed by DNA-sequencing. Transfections were performed using Lipofectamine 3000 reagent according to the manufacturer’s instructions (Invitrogen, Waltham, Massachusetts, USA).
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