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Ha tagged ubiquitin plasmid

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The HA-tagged ubiquitin plasmid is a recombinant DNA construct that expresses a ubiquitin protein with a hemagglutinin (HA) epitope tag. Ubiquitin is a small regulatory protein that can be covalently attached to other cellular proteins, marking them for degradation or other cellular processes. The HA tag allows for the detection and purification of the ubiquitinated proteins using anti-HA antibodies.

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Lab products found in correlation

V5 tagged pcdnacsf3r vector, by Thermo Fisher Scientific (2 mentions) Fugene, by Promega (2 mentions) Aria 2, by BD (2 mentions) Quikchange 2 xl, by Agilent Technologies (2 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions) Pdonr vector, by Thermo Fisher Scientific (1 mentions) Gateway cloning system, by Thermo Fisher Scientific (1 mentions) Prl renilla luciferase vector, by Promega (1 mentions) Ab179808, by Abcam (1 mentions) Ab140601, by Abcam (1 mentions) Ab179434, by Abcam (1 mentions) Magne halotag beads, by Promega (1 mentions) Bp clonase 2, by Thermo Fisher Scientific (1 mentions) Pcdna3.2 v5 dest, by Thermo Fisher Scientific (1 mentions) Halotev protease, by Promega (1 mentions) N ethylmaleimide, by Merck Group (1 mentions) Protease inhibitor, by Roche (1 mentions)

5 protocols using ha tagged ubiquitin plasmid

1

Cloning and Characterization of ETS2 and DUBs

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The open reading frame sequence of ETS2 was cloned into a pDONR vector (Gateway entry vector) and subsequently subcloned into an MYC peptide-tagged expression vector using the Gateway cloning system (Thermo Fisher Scientific; Waltham, MA, USA). The open reading frame sequences of human DUBs were cloned as previously described [19 (link)]. To create a pGL3-LAIR1-p6 vector, an ETS2-responsive luciferase reporter construct, the p6 promoter region of the LAIR1 (Leukocyte Associated Immunoglobulin Like Receptor 1) gene was amplified from HaCaT cell genomic DNA (kindly provided by Dr. Bong Gun Ju at Sogang University) and inserted into a pGL3 luciferase vector, following the methods described in the previous study [20 (link)]. An HA-tagged ubiquitin plasmid was obtained from Addgene (Watertown, MA, USA), and a pRL (Renilla luciferase) vector was purchased from Promega (Madison, WI, USA).
Choi Y., Lee Y., Kim J.S., Zhang P, & Kim J. (2023). USP39-Mediated Non-Proteolytic Control of ETS2 Suppresses Nuclear Localization and Activity. Biomolecules, 13(10), 1475.
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2

Investigating USP2A Interactions with Transcription Factors

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The coding region of human USP2A (accession number NM_004205) without the stop codon was cloned into pcDNA3.2/V5-DEST (Thermo Fisher Scientific) using BP clonase II (Thermo Fisher Scientific). The resultant plasmid encoded C-terminal V5-tagged USP2A. Halo-tagged Oct-1 and Oct-2 expression plasmids were purchased from Promega (Madison, WI). The HA-tagged ubiquitin plasmid was purchased from Addgene (Cambridge, MA). The USP2A, or control pcDNA3.2/V5-DEST plasmid, plasmids encoding Halo-tagged Oct-1 or Oct-2 and HA-tagged ubiquitin were transfected into HEK293FT cells using Lipofectamine 2000 reagent (Thermo Fisher Scientific). The pull-down and elution of Oct proteins were performed using Magne HaloTag beads (Promega) and HaloTEV protease (Promega) as per the manufacturer's instructions. For detection of Western blot bands, corresponding to K48- and K63-linked ubiquitin chains, Oct proteins, HA-tagged ubiquitin, and V5-tagged USP2, 1000-fold-diluted antibodies against polyubiquitin chains formed by K48 residues (ab140601; Abcam) and K63 residues (ab179434; Abcam), Oct-1 (A301-717A; Bethyl Laboratories), Oct-2 (ab179808; Abcam), HA-tag (#3724; CST), and V5-tag (A190-120F; Bethyl Laboratories) were used as the primary antibodies.
Kitamura H., Ishino T., Shimamoto Y., Okabe J., Miyamoto T., Takahashi E, & Miyoshi I. (2017). Ubiquitin-Specific Protease 2 Modulates the Lipopolysaccharide-Elicited Expression of Proinflammatory Cytokines in Macrophage-like HL-60 Cells. Mediators of Inflammation, 2017, 6909415.
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3

Generating CSF3R Mutants via Retroviral Transduction

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CSF3R mutations were generated using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies) as previously described(13 ,15 (link)). Briefly, retrovirus was produced by transfecting HEK293T/17 cells with FuGENE (Promega). After 2 days, the viral vector-containing supernatants were filtered, and infected to Ba/F3 cells followed by flow cytometry (FACS, AriaII, BD Biosciences) sorting of GFP positive cells. Of note, cells with equal intensity of GFP expression were sorted across all samples. For the co-immunoprecipitation (co-IP) assay, an HA-tagged ubiquitin plasmid (#17608, Addgene) was co-transfected with a V5 tagged pcDNACSF3R vector (#12290010, Thermo Scientific).
Zhang H., Schultz A.R., Luty S., Rofelty A., Su Y., Means S., Bottomly D., Wilmot B., McWeeney S.K, & Tyner J.W. (2017). Characterization of the leukemogenic potential of distal cytoplasmic CSF3R truncation and missense mutations. Leukemia, 31(12), 2752-2760.
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4

Generating CSF3R Mutants via Retroviral Transduction

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CSF3R mutations were generated using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies) as previously described(13 ,15 (link)). Briefly, retrovirus was produced by transfecting HEK293T/17 cells with FuGENE (Promega). After 2 days, the viral vector-containing supernatants were filtered, and infected to Ba/F3 cells followed by flow cytometry (FACS, AriaII, BD Biosciences) sorting of GFP positive cells. Of note, cells with equal intensity of GFP expression were sorted across all samples. For the co-immunoprecipitation (co-IP) assay, an HA-tagged ubiquitin plasmid (#17608, Addgene) was co-transfected with a V5 tagged pcDNACSF3R vector (#12290010, Thermo Scientific).
Zhang H., Schultz A.R., Luty S., Rofelty A., Su Y., Means S., Bottomly D., Wilmot B., McWeeney S.K, & Tyner J.W. (2017). Characterization of the leukemogenic potential of distal cytoplasmic CSF3R truncation and missense mutations. Leukemia, 31(12), 2752-2760.
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5

ß2-Adrenergic Receptor Ubiquitination Assay

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ß2-AR ubiquitination was determined by transfecting cells with human influenza hemagglutinin (HA)-tagged ubiquitin plasmid constructs from Addgene (Cambridge, MA, USA): wild type-HA-ubiquitin (Plasmid #17608), Lys 48-HA-Ubiquitin (Plasmid #17605), and Lys 63-HA-Ubiquitin (plasmid # 17606). After 16 hours, cells were treated with MG-132 for 3 hours to induce accumulation of ubiquitinated proteins and harvested in 150 μl lysis buffer containing protease inhibitors (Roche Diagnostics, Germany) and 10 mM deubiquitinating enzyme inhibitor N-ethylmaleimide (Sigma, St. Louis, MO, USA). Five hundred micrograms of protein lysate was pre-cleared before incubation with a ß2-AR antibody for 2 hours at 4°C (El Ayadi et al., 2012 (link)). Immune complexes were recovered using 50 μl protein A-sepharose beads (Pierce, Thermofisher, NY, USA). The beads were washed with lysis buffer, boiled with SDS sample buffer, and separated by SDS-PAGE/Western blot.
Ayadi A.E., Prasai A., Wang Y., Herndon D.N, & Finnerty C.C. (2018). β-Adrenergic receptor trafficking, degradation, and cell-surface expression are altered in dermal fibroblasts from hypertrophic scars. The Journal of investigative dermatology, 138(7), 1645-1655.
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