BMMSCs were seeded on twelve‐well culture dishes and grown to 80%–90% confluence followed by serum starvation for 2h. Cav1.2 (Santa Cruz Biotechnology, sc‐42689) or β‐catenin siRNA (Santa Cruz Biotechnology, sc‐29210) was transfected into BMMSCs at a final concentration of 50 nM, and overexpression plasmid of Cav1.2 (addgene, Plasmid #26572) was transfected into BMMSCs at 500 ng. For the control groups of siRNA or plasmid transfection experiments, scramble siRNA (RiboBio) or control overexpression vector pcDNA6/V5‐His (Invitrogen) was included. Lipo2000 (Invitrogen) was used as a transfection reagent according to the manufacturer's instructions. After transfection, the culture medium was substituted by normal culture medium and cells were harvested at 48 hr for RNA and 72 hr for protein extraction. For detection of the osteogenic differentiation capacity, transfection medium was removed in the next day and replaced by osteogenic induction medium.
Pcdna6 v5 his
Manufactured by Thermo Fisher Scientific
The PcDNA6/V5-His is a mammalian expression vector designed for recombinant protein expression in eukaryotic cells. It features a cytomegalovirus (CMV) promoter for high-level transcription, a V5 epitope tag, and a polyhistidine (6xHis) tag for detection and purification of the expressed protein. The vector also contains a blasticidin resistance gene for selection of stable cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
β catenin sirna, by Santa Cruz Biotechnology (1 mentions)
Scramble sirna, by RiboBio (1 mentions)
Lipo2000, by Thermo Fisher Scientific (1 mentions)
Scrambled rna scrna, by GenePharma (1 mentions)
Pgl3 basic plasmid, by Promega (1 mentions)
Akta purifier, by GE Healthcare (1 mentions)
Ni nta bead, by Qiagen (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Pmoi gfp, by GeneCopoeia (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
8 protocols using pcdna6 v5 his
1
Modulating Osteogenic Differentiation of BMMSCs
2
Generating FOXM1-V5 and ABCG2 Promoter Constructs
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pcDNA6-V5-His was purchased from Invitrogen. pFOXM1 was provided by Dr. Ju-Seog Lee (MD Anderson Cancer Center). To generate the pFOXM1-V5 tag, the coding sequence (CDS) region was amplified by PCR from pFOXM1 DNA. pGL3 basic plasmid was purchased from Promega. To generate the pGL3 basic-ABCG2 promoter, its region from −2105 to +22 was cloned by PCR from human genomic DNA using the indicated primer sets (Table S1 ). Scrambled RNA (scRNA) was purchased from Shanghai GenePharma (Shanghai GenePharma). siFOXM1 was synthesized from ST Pharm Oligo center (ST Pharm). Transfection was carried out according to the manufacturer’s protocol (Jetprime).
3
Protein Expression and Purification of Sirt1, Sirt2, Sirt3, and Sirt6
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Recombinant WT and 2A Sirt1(mouse), F1 fragment (amino acid residues 1–490 of mouse Sirt1), F2 fragment (amino acid residues 491–734 of mouse Sirt1) and deacetylase core (amino acid residues 184–510 of mouse Sirt1) were constructed in the pET15b prokaryotic expression vector by using the Nde1 and Xho1 sites. Prokaryotic expression vectors for human Sirt2, Sirt3 and Sirt6 were kind gift from Dr John M Denu (University of Wisconsin). They were expressed in E. coli, and purified on Ni-NTA beads (Qiagen). All affinity purified proteins were further purified by Superdex 200 HR 10/30 gel-filtration using the AKTA purifier (GE Healthcare). Final preparations of purified proteins were checked by Coomassie staining of SDS polyacrylamide gels. Mammalian expression vectors for human Sirt2 and clover that contain entire coding region were ligated into the NheI and HindIII sites in pcDNA6/V5-His (Invitrogen). Sirt2-CTD and Clover-CTD expression vectors were generated by ligation of the C-terminal region of mouse Sirt1 (amino acid residues 511–734) into the pcDNA6 Sirt2 and Clover expression vectors by using the HindIII and XhoI sites. The Clover-ΔESA expression vector was constructed by sub-cloning the ΔESA Sirt1 CTD into the pcDNA6 Clover construct by using the HindIII and XhoI sites. All constructs were confirmed by DNA sequencing.
4
Expression and Functional Analysis of VHSV P Proteins
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Full-length cDNA of the P genes of ADC-VHS2015-5 (containing P55 in the P protein) and ADC-VHS2012-6 (with the P55L amino acid substitution in the P protein) were amplified from the cDNA of virus-infected HINAE cells using PCR and PCR primers (S4 Table ). The PCR products were ligated into the HindIII/EcoRI sites of pcDNA6/V5-His(Invitrogen) mammalian expression vectors to generate the plasmids pcDNA6-P-wild and pcDNA6-P(P55L). HINAE cells were transfected with those plasmid vectors using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) and treated with blasticidin for 1 week to enrich them. The expression of the P genes in HINAE cells was confirmed by a western blot analysis using an anti-V5 monoclonal antibody (GenWay Biotech Inc.). VHSV P gene–transfected cells were infected with 1 MOI of ADC-VHS2015-5 or ADC-VHS2012-6. At the indicated times after viral infection, the cells were analyzed for their expression of IFN genes and viral RNA using quantitative real-time PCR.
5
Cloning TMPRSS6 and HJV Isoforms
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The cDNAs encoding TMPRSS6‐1 and HJV were obtained and cloned as previously described.26 TMPRSS6‐2 construct was obtained using the QuikChange site‐directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). TMPRSS6‐3 and TMPRSS6‐4 constructs were obtained by inserting a synthetic double‐stranded DNA block coding for the isoform sequence into a modified form of pcDNA6/V5‐His (Invitrogen) that was previously described.26 Additional details are provided in the supplemental data.
6
ZIKV Nonstructural Protein Expression in Mammalian Cells
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pMOI-GFP and pMOI-PARP11 (Homo sapiens) expression plasmids were purchased from GeneCopoeia and described previously [34 (link)]. The viral RNA of the GZ01/2016 strain was isolated and used in reverse transcription PCR experiments to obtain the complementary DNA (cDNA) sequence of ZIKV nonstructural NS1 and NS3 proteins. ZIKV NS1 and NS3 genes were cloned into the pcDNA6/V5-His expression vector (Invitrogen) using standard molecular techniques and verified by sequencing. DsRed-PARP11, EGFP-PARP12, Flag-PARP11, HA-PARP12, HA-PARP12 ZnF, HA-PARP12 WWE, HA-PARP12 PARP, EGFP-PARP11 WWE domain, EGFP-PARP11 PARP domain, YFP-PARP11, Flag-PARP13 and GFP-PARP11 mutants were cloned using standard molecular cloning and oligonucleotide mutagenesis methods. To create a stable cell line for PARP11 expression, PARP11 was cloned into the pMXsIG-IgkFLAG vector and co-transfected into HEK293T cells with VSV glycoprotein and pCpG helper plasmids. 48 h after transfection, the culture supernatant was collected and added into WT or PARP12−/− A549 cells for infection. The cells were collected 72 h after infection, and the PARP11-overexpressing cells were then sorted by fluorescence-activated cell sorting (FACS).
7
CTNS-LKG Fusion Protein Expression
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The full-length CTNS-LKG coding sequence was fused in frame at its carboxyl-terminal region with RFP cDNA and inserted into the expression vectors pcDNA 3.1 (Invitrogen). Alternatively, the full-length cDNA encoding cystinosin-LKG was subcloned into pcDNA6/V5His (Invitrogen) or pEGFP-N1 (Clontech) vectors. S396A, S397A, L398A, K399A, G400A, ΔYFPQA and ΔSSLKG deleted mutants were generated by site-directed mutagenesis with Q5 Site-Directed Mutagenesis Kit (New England Biolabs). Sequence integrity was tested by direct sequencing. After plasmid purification, cells were transfected in HK-2 cells using Lipofectamine LTX (Invitrogen), following manufacturer’s instructions. Cells stably overexpressing RFP fusion protein were selected with 3 μg/ml hygromycin B.
8
Construction and Characterization of FAD Mutant Library
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The backbone plasmid pcDNA6/V5-His was purchased from Invitrogen Inc. (Carlsbad , CA). The APP695 cDNA (courtesy of Dr. G. Levesque, CHUQ, Quebec) was inserted by ligation between Kpn1 and Xba1 cut sites. The ensuing plasmid was then mutated using the New England Biolabs (NEB, Ipswich, MA) mutagenesis Q5 kit in 29 different reactions. The 29 new plasmids each represented a form of FAD and served as a “normal” library version of each FAD. The mutations were located in exons 16 and 17 to better demonstrate the protective effects of A673T. Another “mutated” library was created by adding an additional A673T mutation to each FAD plasmid. Prior to the start of the experiments, the plasmids underwent Sanger sequencing to ensure that the only mutations present were those under study.
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