Pcdna3.1 v5 his a

Manufactured by Thermo Fisher Scientific

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The PcDNA3.1/V5-His A is a plasmid vector designed for the expression of recombinant proteins in mammalian cell lines. It provides a strong cytomegalovirus (CMV) promoter for high-level expression and a C-terminal V5 epitope and polyhistidine (6xHis) tag for detection and purification of the expressed protein.

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11 protocols using pcdna3.1 v5 his a

Cloning and Expression of FFAR Receptors

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The myristoylated human Akt1 (myr-Akt) was cloned into pcDNA3 vector or the empty pcDNA3 expression vector as control. The coding regions (CDS) of human FFAR3 (NM_005304), human FFAR2 (NM_005306), mouse Ffar3 (NM_001033316), and mouse Ffar2 (NM_001168509) were cloned into the pcDNA3.1/V5-His A (Invitrogen) expression vector.

Ang Z., Er J.Z., Tan N.S., Lu J., Liou Y.C., Grosse J, & Ding J.L. (2016). Human and mouse monocytes display distinct signalling and cytokine profiles upon stimulation with FFAR2/FFAR3 short-chain fatty acid receptor agonists. Scientific Reports, 6, 34145.

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Reconstitution of MARCKS Expression

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The reconstitution of MARCKS expression in MARCKS KO cells was performed using the expression plasmid pcDNA 3.1 V5H6-A MARCKS-WT consisting of the full length human MARCKS coding sequence (amplified using the primers 5′-TCGAATTCATGGGTGCCCAGTTCT-3′ and 5′-TGGATCCTCCCTCTGCCGCCTCC-GCT-3′) cloned into the vector pcDNA 3.1/V5-His A (Invitrogen) via the restriction sites EcoRI and EcoRV.

Huber R., Diekmann M., Hoffmeister L., Kühl F., Welz B, & Brand K. (2022). MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type. Antioxidants, 11(8), 1600.

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Molecular Cloning of Plasmid Constructs

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pcDNA3.1-v5-hYSK1 and deletion fragments of hYSK1 were generated by PCR using the human cDNA clone-hYSK1 (Origene Technologies, MD, USA) as a template. The PCR product was purified, digested with EcoRI/XhoI, and cloned into the EcoRI/XhoI sites of pcDNA3.1-v5-HisA (Invitrogen, MA, USA). For TOPO-GFP-hYSK1, the PCR product was inserted into the pcDNA3.1-NT-GFP-TOPO vector (Invitrogen, MA, USA). The GST-p16^INK4a and GST-deletion fragments of p16^INK4a were inserted in-frame into the BamHI/XhoI site of the pGEX-5X-1 vector (Amersham Biosciences, PA, USA). For pDsRed-p16^INK4a, the PCR product was cloned into the ApeI site of the pDsRed vector (Clontech Laboratories, CA, USA). HA-SP-1 was cloned into the EcoRI/XhoI site of the pCMV-HA and pCMV-Myc vectors (Clontech Laboratories, CA, USA). The shRNA-hYSK1 plasmid was constructed into the pSilencer 4.1-CMV-hyg vector (Ambion, NY, USA). The pGL2-p16-luc vector was a gift from Dr. Gordon Peters (Cancer Research UK, London) and the pGL2-MMP-2-luc vector was a gift from Dr. Etty N. Benveniste (The University of Alabama at Birmingham, Birmingham, AL). Various expression vectors were amplified in E. coli XL1-blue or BL21 cells and plasmids were purified using a Qiagen midi kit (Qiagen, Hilden, Germany). The DNA sequences of all plasmids were confirmed by sequencing (Dye Terminator ABI Type Seq., Bionex, NJ, USA)

Lee M.H., Dong Z., Surh Y.J, & Choi B.Y. (2017). hYSK1 promotes cancer cell proliferation and migration through negative regulation of p16INK4a under hypoxic conditions. Oncotarget, 8(51), 89072-89085.

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Cloning and Mutagenesis of Chemokine Receptors

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The complementary DNAs (cDNAs) encoding the C-C chemokine receptors and CXC-receptors (R&D, Minneapolis, MN, USA) were cloned into the expression vector pcDNA3.1/V5-His-A (Invitrogen) at the BamHI and XholI sites. Single mutants were constructed by PCR-based site-directed mutagenesis. CHO-K1 cells were seeded onto 96-well, poly-d-lysine-treated cell culture plates (PerkinElmer) at a density of 2.7 × 10⁴ cells per well. After overnight culture, the cells were transiently transfected with plasmid DNA using Lipofectamine 2000 transfection reagent (Invitrogen).

Yao W., Ba Q., Li X., Li H., Zhang S., Yuan Y., Wang F., Duan X., Li J., Zhang W, & Wang H. (2017). A Natural CCR2 Antagonist Relieves Tumor-associated Macrophage-mediated Immunosuppression to Produce a Therapeutic Effect for Liver Cancer. EBioMedicine, 22, 58-67.

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Cloning and Transfection of CYP3A43 in Cells

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The CYP3A43 expression plasmid pcDNA3.1-V5-His A-CYP3A43 was obtained from IGE BIOTECHNOLOGY LTD. The vector plasmid pcDNA3.1/V5-His A was purchased from Invitrogen. The short-hairpin RNA targeting human CYP3A43 (Forward oligo
5′-CCGGGCCTGGTACTCCTCTATATTTCTCGAGAAATATAGAGGAGTACCAGGCTTTTTG-3′
Reverse oligo
5′-AATTCAAAAAGCCTGGTACTCCTCTATATTTCTCGAGAAATATAGAGGAGTACCAGGC-3′) was synthesized by IGE BIOTECHNOLOGY LTD, and then incorporated into a pLKO.1 TRC cloning vector (Cat. #10878, Addgene) following the protocol provided by the manufacturer.
The transfections were performed with PEI reagent according to the manufacturer’s instructions. The trypsinized single cells in a medium with 10% FBS were transfected with plasmids or shRNA and cultured for 24 h before the cells were used for subsequent analysis.

Wei Q.Y., Lau A.T., Mo H.Y., Zhong Q.H., Zhao X.Y., Yu F.Y., Han J., Wu Y.Y, & Xu Y.M. (2022). Effects of CYP3A43 Expression on Cell Proliferation and Migration of Lung Adenocarcinoma and Its Clinical Significance. International Journal of Molecular Sciences, 24(1), 113.

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Analyzing VWC2 Interactions with TGF-β Superfamily

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To determine the interaction between VWC2 and TGF-β superfamily members, 293 cells were plated and transfected with pcDNA3 -Vwc2-HA together with an empty pcDNA3.1-V5/His A (Invitrogen), pcDNA3.1-Bmp-2-V5/His, pcDNA3.1-Bmp-4-V5/His, pcDNA3.1-Bmp-6-V5/His, pcDNA3.1-Bmp-7-V5/His, pcDNA3.1-Tgf-β1-V5/His, pcDNA3.1-Tgf-β2-V5/His, pcDNA3.1-Tgf-β3-V5/His, pcDNA3.1 -Activin-βA-V5/His, or pcDNA3.1-Activin-βB-V5/His vector using FuGENE6 transfection regent. The binding assay was performed by immunoprecipitation (IP) with the conditioned media collected from the transfected cells and Western blot (WB) analysis as previously described [17 (link)].

Almehmadi A., Ohyama Y., Kaku M., Alamoudi A., Husein D., Katafuchi M., Mishina Y, & Mochida Y. (2018). VWC2 Increases Bone Formation Through Inhibiting Activin Signaling. Calcified tissue international, 103(6), 663-674.

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DNA-Protein Interaction Analysis via EMSA

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The experiment was performed with LightShift^® Chemiluminescent EMSA kit (Thermo Fisher Scientific, catalog no. 20148), following the manufacturer’s instructions. Full length of E2F1 or MYC was cloned into vector pcDNA3.1/V5-HisA (Invitrogen, catalog no. V81020). Briefly, double-stranded biotin-labeled consensus DNA was incubated with HEK293T cell nuclear extract with ectopically expressed E2F1 or MYC in a 1× binding buffer, 50 ng/µl poly(dI:dC)·poly(dI:dC), 2.5% glycerol, 0.05% NP-40, 5 mM MgCl₂, and 10 mM EDTA, and 200-fold excess of unlabeled probes was used for competition assays. The protein complexes were resolved on 6% DNA retardation gels for 1 h at 100 V, transferred to Biodyne B Nylon Membranes (PALL, catalog no. 60208), cross-linked, and detected using streptavidin-HRP conjugate and a chemiluminescent substrate. The oligos used are listed in Supplementary Table 3.

Dong X., Zhang Q., Hao J., Xie Q., Xu B., Zhang P., Lu H., Huang Q., Yang T., Wei G.H., Na R, & Gao P. (2021). Large Multicohort Study Reveals a Prostate Cancer Susceptibility Allele at 5p15 Regulating TERT via Androgen Signaling-Orchestrated Chromatin Binding of E2F1 and MYC. Frontiers in Oncology, 11, 754206.

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Molecular Tools for Transcriptional Regulation

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Genomic ORF50 (pcDNA3-G50) was cloned into pcDNA3 as described in (Lukac et al., 1998 (link)) and expresses the full length Rta protein. Mouse Notch intracellular domain (NICD) 1 was cloned into p3xFLAG-CMV-7 expression vector (Sigma) (Ong et al., 2006 (link)) and generously given to our lab by Dr. Raphael Kopan. HDAC1-FLAG expresses human HDAC1 fused to the FLAG epitope tag (a gift of Eric Verdin; (Shin et al., 2014 (link))). H2B-mCherry was a gift from Robert Benezra (Addgene plasmid # 20972) (Nam and Benezra, 2009 (link)). RtaΔSTAD (Lukac et al., 1999 (link)) is a C-terminal truncation mutant of Rta in which amino acids 530–691 of the cognate protein are deleted. The mutant protein lacks Rta’s transcriptional transactivation domain, so is incapable of transactivating viral promoters or reactivating the virus in PEL latency models. RtaΔSTAD was cloned as a C-terminal fusion to the V5 epitope tag in the vector pcDNA3.1/V5-His A (Invitrogen) to permit detection by anti-V5 antibodies. pcDNA3 (Invitrogen) was used as an empty vector control for all experiments.

DeCotiis J.L., Ortiz N.C., Vega B.A, & Lukac D.M. (2017). An easily transfectable cell line that produces an infectious reporter virus for routine and robust quantitation of Kaposi’s sarcoma-associated herpesvirus reactivation. Journal of virological methods, 247, 99-106.

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Generation of GRTH-expressing COS-1 Cell Line

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COS-1 cells were obtained from ATCC CRL-1650 and cultured in a T75 flask at 37 °C with 5% CO₂, containing Dulbecco modified Eagle medium (DMEM) high glucose, GlutaMax (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Inc., Lawrenceville, GA) and 1× antibiotic–antimycotic (#15240062, Thermo Fisher Scientific). The full-length human GRTH cDNA fragment (GenBank Acc # AF155140) was cloned into pcDNA3.1/V5-His A (Thermo Fisher Scientific, Waltham, MA) at KpnI and XbaI restriction sites, and the sequence was confirmed. This GRTH plasmid construct and COS-1 cells were used to generate a stable cell line expressing human GRTH. The expression of pGRTH in a western blot was assessed using a custom-made affinity-purified phospho-site-specific GRTH polyclonal antibody raised in rabbit to the peptide sequence (CKLIDL[pT239]KIRV) of human GRTH.

Raju M., Kavarthapu R., Anbazhagan R., Hassan S.A, & Dufau M.L. (2021). Blockade of GRTH/DDX25 Phosphorylation by Cyclic Peptides Provides an Avenue for Developing a Nonhormonal Male Contraceptive. Journal of medicinal chemistry, 64(19), 14715-14727.

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Cloning of Human MLKL cDNA

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Human MLKL cDNAs were amplified by PCR using human MLKL pFN21A HaloTag®CMV Flexi®Vector (Kazusa DNA Research Institute, Kisarazu, Japan). The PCR products and pcDNA3.1/V5-His A (ThermoFisher Scientific) were digested with EcoRI/XhoI and each fragment was ligated, forming human MLKL pcDNA3.1/V5-His A vectors.

Zhang X., Kitatani K., Toyoshima M., Ishibashi M., Usui T., Minato J., Egiz M., Shigeta S., Fox T., Deering T., Kester M, & Yaegashi N. (2017). Ceramide nanoliposomes as a MLKL-dependent, necroptosis-inducing, chemotherapeutic reagent in ovarian cancer. Molecular cancer therapeutics, 17(1), 50-59.

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