Ha ub

Manufactured by Addgene

Sourced in United States

HA-Ub is a recombinant protein consisting of the ubiquitin (Ub) protein fused to the haemagglutinin (HA) epitope tag. It is used as a research tool to study ubiquitin-related processes in cells.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions) Ha k48, by Addgene (3 mentions) Ha ub k48, by Addgene (2 mentions) Ha ub k33, by Addgene (2 mentions) Ha ub k63, by Addgene (2 mentions) Ha k63, by Addgene (2 mentions) Transit lt1 transfection reagent, by Mirus Bio (2 mentions) Quickchange 2 xl site directed mutagenesis kit, by Agilent Technologies (2 mentions) Bcl 6 clone id, by Thermo Fisher Scientific (2 mentions) Ha p110, by Addgene (2 mentions) Pcdna3.1 myc his b, by Thermo Fisher Scientific (1 mentions) Calcein, by Thermo Fisher Scientific (1 mentions) Cycloheximide (chx), by Cell Signaling Technology (1 mentions) Flag tag (flag), by Santa Cruz Biotechnology (1 mentions) Col10a1, by Thermo Fisher Scientific (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Bromodeoxyuridine (brdu), by Santa Cruz Biotechnology (1 mentions) P taz, by Cell Signaling Technology (1 mentions) Edta free cocktail inhibitor tablets, by Thermo Fisher Scientific (1 mentions) P last1, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

PRK5-HA-Ub-K48 (7 citations) PRK5-HA-Ub-K63 (6 citations) PRK5-HA-Ub-WT (6 citations) PRK5-HA-Ub (5 citations) HA-Ub-K0 (3 citations) HA-Ub-K48 (3 citations) HA-Ub-K63 (3 citations) HA-Ub (K33) (2 citations) PcDNA3.1-HA-Ub (2 citations) HA tagged Ub (2 citations)

16 protocols using ha ub

Engineered Plasmids for Cell Studies

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We mainly used pcDNA3.1 myc-His (+) B (V80020; Invitrogen) and pHR_EF1a (modified from pHR_PGK, #79120; Addgene) for transit transfection and virus packing, respectively. Human MARCH5 (including its variants), PEX3 (including its variants), PEX19 (including its variants), and PMP70 were cloned into pcDNA3.1 with a C-terminal Myc or V5 or triple FLAG tag using Gibson assembly. MARCH5-mCherry-Myc (including its variants) and PEX3-YFP (including its variants) were cloned into pcDNA3.1 and pHR_EF1a. mCherry-SKL, RFP-SKL, GFP-SKL, and GFP-RFP-SKL were cloned into pHR_EF1a. HA-Ub was purchased from Addgene (#18712). All plasmids were confirmed by Sanger sequencing.

Zheng J., Chen X., Liu Q., Zhong G, & Zhuang M. (2021). Ubiquitin ligase MARCH5 localizes to peroxisomes to regulate pexophagy. The Journal of Cell Biology, 221(1), e202103156.

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Plasmid construction and shRNA targeting

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HA-Ub (WT), HA-Ub (K6) only, HA-Ub (K11) only, HA-Ub (K27) only, HA-Ub (K29) only, HA-Ub (K33) only, HA-Ub (K48) only, HA-Ub (K63) only, and HA-GASC1 were purchased from Addgene. HA-Ub (K63R) was generated by cloning the corresponding cDNAs into the pCMV-HA vector via XbaI/NotI sites. pCMV-Myc-ROCK2, pCMV-Myc-ROCK2-CAT (aa 5-553), and pCMV-Myc-ROCK2-RB/PH (aa 686-1388) were generated by subcloning the corresponding cDNAs into the pCMV-Myc vector via KpnI/XhoI or XbaI/NotI sites. HA-FBXO42 and HA-FBXO42-△F-box (deleting aa 44-93) was generated by subcloning the corresponding cDNAs into the pCMV-HA vector via XhoI/BamHI sites. pCMV-Myc-ROCK2-K121R was generated using the Quick Change Q5 Site-Directed Mutagenesis Kit (NEBaseChanger) according to the manufacturer’s instructions. The pLKO.1-puro lentiviral MISSION shRNA constructs targeting endogenous GASC1 (shGASC1 CDS1 (TRCN0000022056): sense, 5′-GCCTCTGACATGCGATTTGAA-3′, and shGASC1 CDS2 (TRCN0000022055): sense, 5′-CCTTGCATACATGGAGTCTAA-3′), FBXO42 (shFBXO42 CDS (TRCN0000134822): sense, 5′-CCATCAGTGTTATCATGGTTT-3′, and a non-targeting (NT) control shRNA (TRC1/1.5) were from Sigma-Aldrich. Details of plasmid construction are available upon request.

Shao N., Cheng J., Huang H., Gong X., Lu Y., Idris M., Peng X., Ong B.X., Zhang Q., Xu F, & Liu C. (2021). GASC1 promotes hepatocellular carcinoma progression by inhibiting the degradation of ROCK2. Cell Death & Disease, 12(3), 253.

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Trp53, LAST1 and TAZ Regulation

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Antibodies against Trp53 (#2524; dilution 1:1000), LAST1 (#3477; dilution 1:1000), pLAST1 (#8654; dilution 1:1000), TAZ (#83669; dilution 1:1000), pTAZ (#59971; dilution 1:1000), HA (#5017; dilution 1:1000), GAPDH (#5174; dilution 1:1000); cycloheximide (CHX) and insulin-transferrin-sodium selenite media supplement (ITS Supplement) were purchased from Cell Signaling Technology. BrdU (#sc-32323; dilution 1:1000), β-TrCP (#sc-390629; dilution 1:1000), GFP (#sc-9996; dilution 1:1000) and flag (#sc-7945; dilution 1:1000) antibodies were obtained from Santa Cruz Biotechnology. Col10a1 (#14-9771-80; dilution 1:1000) antibody, EDTA-free cocktail inhibitor tablets, calcein and BrdU labeling were obtained from Fisher Scientific™. The Transfection Reagent FuGENE^® HD was ordered from Promega (USA). The plasmids GFP-Trp53, flag-TAZ, and HA-Ub were from Addgene (USA).

Li Y., Yang S.T, & Yang S. (2022). Trp53 controls chondrogenesis and endochondral ossification by negative regulation of TAZ activity and stability via β-TrCP-mediated ubiquitination. Cell Death Discovery, 8, 317.

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Plasmid Transfection for EIF3H Study

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The wild-type and mutant constructs of EIF3H plasmids were purchased from the Hanbio Biotechnology Co., Ltd. (Shanghai, China). The HA-K48, HA-K63, and HA-Ub plasmids were acquired from Addgene. The small interfering RNAs targeting EIF3H (5ʹ-GCAACTCTTGGAAGAAATATA-3ʹ) and (5ʹ-CCCAAGGATCTCTCTCACTAA-3ʹ) were gained from Ruibo Biotechnology Co., Ltd. (Guangzhou, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was utilized for plasmid and siRNA transfection according to the manufacturer's recommendations.

Tang J., Long G., Li X., Zhou L., Zhou Y, & Wu Z. (2023). The deubiquitinase EIF3H promotes hepatocellular carcinoma progression by stabilizing OGT and inhibiting ferroptosis. Cell Communication and Signaling : CCS, 21, 198.

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Plasmids and siRNAs for Ubiquitin Research

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The The full-length and deletion mutant constructs of TAZ and USP26 were obtained from Hanbio Biotechnology Co. Ltd. (Shanghai, China). The HA-K6, HA-K11, HA-K27, HA-K29, HA-K33, HA-K48, HA-K63, and HA-Ub plasmids were acquired from Addgene. Small interfering RNAs targeting USP26 (siRNA-1: 5′-GCACAAGACUUCCGUUGGA-3′; 5′-AAACAGAUCUGGUUCACUU-3′) were synthesized by Genepharma (Shanghai, China).

Tang J., Luo Y, & Xiao L. (2022). USP26 promotes anaplastic thyroid cancer progression by stabilizing TAZ. Cell Death & Disease, 13(4), 326.

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Cloning and Characterization of OPN-i and Bcl-6 Constructs

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OPN-i expression vectors, pMLS5, OPN-i–Flag, and OPN-i–GFP were described previously ^{17 (link)}. A tandem HA-Flag tag was introduced at the C-terminus of OPN-i cDNA by PCR using primers containing BamHI and EcoRI sites followed by cloning into pBABE-GFP vector. Bcl-6 cDNA was obtained from Open Biosystems (Bcl-6 Clone ID: 6309948), sequenced in full, before complete coding region sequences were cloned inframe with a Flag tag at the N-terminus into retroviral expression vector MSCV-IRES-GFP. The following plasmids were obtained from Addgene: p85α (plasmid 1399 and 1407), HA-p110 (plasmid 12522 and 15691) ^{50 (link), 51 (link)} and HA-Ub (plasmid 17608) ^{52 (link)}. Deletion constructs of Bcl-6, Flag–Bcl-6, and OPN-i Y166F mutants were generated by PCR-mediated mutagenesis with the QuickChange II XL Site-Directed Mutagenesis Kit (Agilent). The accuracy of all plasmids was confirmed by DNA sequencing. Retroviral stocks were generated by transfection of 293T cells with plasmids expressing pBABE-GFP control or pBABE-GFP plus OPN-i wild-type or OPN-i Y166F along with pCL-Eco packaging vector using TransIT-LT1 transfection reagent (Mirus). Viral supernatants were collected 72 h later before infection of CD4⁺ T cells, as described below.

Leavenworth J.W., Verbinnen B., Yin J., Huang H, & Cantor H. (2014). A p85α–osteopontin axis couples the ICOS receptor to sustained Bcl-6 expression by follicular helper and regulatory T cells. Nature immunology, 16(1), 96-106.

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Plasmid Transfection for Ubiquitination Analysis

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The mammalian expression plasmids Flag-tagged TBK1 and V5-tagged SRA were stored in our laboratory. Flag-CYLD, Flag-TBK1 KD, ULD, SDD, CTD, 30R, 401R, and 30R/401R were obtained from BGI (BGI group, Shenzhen, China). HA-Ub, HA-Ub-K48, HA-Ub-K63, and HA-Ub-K33 were purchased from Addgene (http://www.addgene.org). The HEK293T cells were cultured in DMEM high glucose supplemented with 10% FBS. Cells were seeded in 10-cm dishes and were transfected with 10 μg plasmid with PEI (Polysciences, Warrington, PA, USA) according to the manufacturer’s instruction.

Li L., Luo J., Zhu Z., Xu Q., Wang P., Chang B., Wang D., Yu L., Lu X., Zhou J., Zuo D, & Chen Q. (2022). SRA Suppresses Antiviral Innate Immune Response in Macrophages by Limiting TBK1 K63 Ubiquitination via Deubiquitinase USP15. Microbiology Spectrum, 10(6), e02028-22.

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Cloning and Characterization of OPN-i and Bcl-6 Constructs

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Transfecting Src, HIF-1α, and Ubiquitin

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HA-HIF-1α, HA-Ub, and pcDNA3-Src plasmids were purchased from Addgene (Watertown, MA, USA). Small interfering RNAs (siRNAs) targeting Src were purchased from Santa Cruz Biotechnology. Cells were transiently transfected with plasmids or siRNAs using Lipofectamine 2000 or RNA iMAX reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Jo M.J., Kim B.G., Kim W.Y., Lee D.H., Yun H.K., Jeong S., Park S.H., Kim B.R., Kim J.L., Kim D.Y., Lee S.I, & Oh S.C. (2021). Cannabidiol Suppresses Angiogenesis and Stemness of Breast Cancer Cells by Downregulation of Hypoxia-Inducible Factors-1α. Cancers, 13(22), 5667.

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Molecular Interactions in USP11 and Sirt3 Study

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Human Flag-USP11, Flag-USP11-C318S, HA-Ubiquitin (Ub) and HA-Ub mutants were purchased from Addgene (MA, USA). The protein information of USP11 (UniProt ID: P51784) and Sirt3 (UniProt ID: Q9NTG7) was acquired from UniProt website (https://www.uniprot.org/). Then the coding sequence (CDS) of USP11 (GenBank ID: BC140849.1) and Sirt3 (GenBank ID: AF083108.2) was obtained from National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/). The full-length sequence or different domain sequences of USP11 and Sirt3 were synthesized and subcloned into Flag-, or Myc-tagged pEX-3 (pGCMV/MCS/Neo) vectors by GenePharma Biotechnology Co., LTD (Shanghai, China). Human NP cells or Human embryonic kidney T cells (HEK 293 T cells, purchased from QuickCell, China) were transfected with above plasmid using Lipo8000™ reagent (C0533, Beyotime, China). Briefly, the day before transfection, the cells were seeded in 6-well plates (about 5 × 10⁴ cells per well). The culture medium was changed when the cells reached 80% confluence. Added the mixture with the amount of 130 μl (125 μl of Opti-MEM + 2.5 μg of DNA + 4 μl of Lipo8000™ reagent) into each well, and incubated for 24 h.

Zhu J., Sun R., Sun K., Yan C., Jiang J., Kong F, & Shi J. (2023). The deubiquitinase USP11 ameliorates intervertebral disc degeneration by regulating oxidative stress-induced ferroptosis via deubiquitinating and stabilizing Sirt3. Redox Biology, 62, 102707.

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