Prk5 ha ubiquitin k63

Manufactured by Addgene

Sourced in United States

PRK5-HA-Ubiquitin-K63 is a molecular biology tool used for studying ubiquitination, a post-translational modification process. It consists of a ubiquitin molecule with a lysine-63 (K63) linkage, tagged with a hemagglutinin (HA) epitope, and expressed under the control of the PRK5 promoter.

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Close spelling variants of this product

HA-Ubiquitin (37 citations) PRK5-HA-ubiquitin (11 citations) HA-K63 (4 citations) Ubiquitin (3 citations) HA-ubiquitin-K63 (2 citations)

15 protocols using prk5 ha ubiquitin k63

Ubiquitination Detection in Immunoprecipitates

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For immunoprecipitation, cells were lysed by sonication in lysis buffer as described previously49 (link). Unless otherwise noted, 50 μg total protein lysate was loaded onto gel. For immunoprecipitation with anti-CUGBP1 or ezrin antibody, 1 mg protein lysates were incubated with 1 μg relevant antibody overnight at 4 °C, followed by incubation with protein A or protein G for 1 h at 4 °C. The expression vector for wild-type UB pCMV-6His-HA-Ubiquitin was obtained from Dr Antonio Iavarone, Columbia University, as a kind gift. The expression vector for UB mutant pRK5-HA-Ubiquitin-KO (17603), pRK5-HA-Ubiquitin-K11 (22901), pRK5-HA-Ubiquitin-K48 (17605), pRK5-HA-Ubiquitin-K29R (17602) and pRK5-HA-Ubiquitin-K63 (17606) plasmids were purchased from Addgene. Plasmid transfection was conducted with the Lipofectamine 2000 reagent from Invitrogen (11668-019, Carlsbad, CA, USA), according to the manufacturer's protocol. For CUGBP1 or ezrin immunoprecipitation for ubiquitination detection, cells were treated with 10 μM MG132 (American Peptide, Sunnyvale, CA, USA) for 90 min at 72 h post-transfection. Half of the immunoprecipitates were loaded onto one gel, gel was transferred onto nitrocellulose membrane as usual, then prepared for Ubiquitin blotting by boiling the membrane for 10 min.

Liu X., Zhao B., Sun L., Bhuripanyo K., Wang Y., Bi Y., Davuluri R.V., Duong D.M., Nanavati D., Yin J, & Kiyokawa H. (2017). Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes. Nature Communications, 8, 14286.

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Ubiquitin Conjugation and Knockdown Assay

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pRK5-HA-Ubiquitin-K48 (#17605; RRID: Addgene_17604), HA-Ubiquitin (#18712; RRID: Addgene_18712), pRK5-HA-Ubiquitin-K63 (#17606; RRID:Addgene_17606), Rc/CMV Cyclin D1 HA (#8948) plasmids were purchased from addgene. pSG5HA-DZIP3 and pSG5FLAG-DZIP3 were described previously (17 (link)). Flag-DZIP3 and Flag-DZIP3 deletion constructs were cloned in gateway cloning vectors as per standard protocol (Invitrogen). For transient knockdown, cells were transfected by electroporation using the Neon transfection system (Invitrogen) and also using INTERFERin (Polyplus) or RNAimax (Invitrogen) as per the manufacturer's instruction. For transient overexpression, Lipofectamine 2000 (Invitrogen) and CALPHOS (Clontech) were used according to the manufacturer's instructions.

Kolapalli S.P., Sahu R., Chauhan N.R., Jena K.K., Mehto S., Das S.K., Jain A., Rout M., Dash R., Swain R.K., Lee D.Y., Rusten T.E., Chauhan S, & Chauhan S. (2020). RNA binding RING E3-ligase DZIP3/hRUL138 stabilizes Cyclin D1 to drive cell cycle and cancer progression. Cancer research, 81(2), 315-331.

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LMP1 Variants and Signaling Pathways

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The plasmids pCMV-HA-LMP1 wildtype, pCMV-HA-LMP1(AAA/Δ371–386) harboring a P204xQxT to AxAxA mutation within CTAR1 and lacking the 16 C-terminal amino acids of CTAR2, pCMV-HA-LMP1(AAA/Y384G), pSV-LMP1, pSV-LMP1(Y384G), pcDNA3-Flag-IKK2, and pRK5-HA-JNK1 have been described^{16 (link),49 (link)}. The vector pSV-NGFR-LMP1 encoding a fusion protein of aa 1–279 of human low affinity p75 NGF-receptor and aa 196–386 of LMP1 has been described^{17 (link),48 (link)}. pCMV5-TPL2wt.MT (provided by C. Patriotis) and pcDNA3-Flag-p105 (provided by D. Krappmann) have been described^{69 (link),70 (link)}. The vector pEF4C-3xFlag-IKKγwt (NEMO) was a kind gift of D. Krappmann. pRK5-HA-Ubiquitin K63 (all lysines mutated to arginines except of K63) was obtained from Addgene and has been described^{71 (link)}.

Voigt S., Sterz K.R., Giehler F., Mohr A.W., Wilson J.B., Moosmann A, & Kieser A. (2020). A central role of IKK2 and TPL2 in JNK activation and viral B-cell transformation. Nature Communications, 11, 685.

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Cloning and Mutating Monkey MAVS Gene

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The monkey MAVS gene was amplified from Vero cells with gene-specific primers based on MAVS sequences available in the GenBank database (accession no. NM_001042666, Table 1) and subcloned into p3×FLAG-CMV (Sigma, E7658) to generate the following expression plasmid: pFLAG-MAVS. The following recombinant plasmids of the truncated MAVS gene were constructed: p3×FLAG-CMV-MAVS1, MAVS2, and MAVS3 (aa 1–201, aa 202–359, and aa 360–541, Table 1). FLAG-MAVS3 mutants were generated by site-directed mutagenesis (Rui Biotech Co., Ltd.): FLAG-MAVS3mt1, FLAG-MAVS3mt2, FLAG-MAVS3mt3, FLAG-MAVS3mt4, FLAG-MAVS3mt5 (K363A, K372A, K421A, K462A, and K501A, respectively) and FLAG-MAVS3mt-sim (K363A, K462A, and K501A, simultaneously). All of the above plasmids were confirmed to be correct by sequencing. pRK5-HA-ubiquitin (17608), pRK5-HA-ubiquitin-K48 (17605), and pRK5-HA-ubiquitin-K63 (17606) were purchased from Addgene.

Hou L., Hu X., Guo J., Quan R., Wei L., Wang J., Song J, & Liu J. (2021). Avian Metapneumovirus Subgroup C Induces Mitochondrial Antiviral Signaling Protein Degradation through the Ubiquitin-Proteasome Pathway. Viruses, 13(10), 1990.

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Ubiquitin Plasmid Toolkit

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Plasmids pRK5-HA-Ubiquitin-WT, pRK5-HA-Ubiquitin-K48, and pRK5-HA-Ubiquitin-K63 were purchased from Addgene (Watertown, MA, USA). Plasmid pRK5-HA-Ubiquitin-K48R/K63R was a generous gift from Dr. Guang-Chao Chen (Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan) [39 (link)].

Jan Fada B., Guha U., Zheng Y., Reward E., Kaadi E., Dourra A, & Gu H. (2023). A Novel Recognition by the E3 Ubiquitin Ligase of HSV-1 ICP0 Enhances the Degradation of PML Isoform I to Prevent ND10 Reformation in Late Infection. Viruses, 15(5), 1070.

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Plasmid Construction and Lentiviral Transduction

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Human RNF166, AMOT and AMOTL2 expression plasmids in the pENTER vector were purchased from Vigene Biosciences (CH893837, CH808629 and CH821284), and pCMV-flag YAP2-5SA [43 (link)] was a gift from Kunliang Guan (Addgene plasmid #27371). The human TNKS plasmid was generated by PCR amplification of the corresponding cDNAs and cloning into pcDNA 3.1. The indicated plasmids were subcloned into pLV-EF1α-MCS-IRES-Puro, pLV-EF1α-MCS-IRES-Bsd, pcDNA 3.1 or pGEx-6P1 expression vectors with various tags, depending on the experimental purposes. The lentiviral packaging plasmids pMD2.G (#12259) and psPAX2 (#12260) and ubiquitin-related plasmids pRK5-HA-Ubiquitin-WT (#17608), pRK5-HA-Ubiquitin-K48 (#17605), and pRK5-HA-Ubiquitin-K63 (#17606) were obtained from Addgene.
RNF166 shRNA was generated according to the Addgene’s pLKO.1-TRC cloning vector protocol. The targeting sequences of RNF166 were as follows: #1 sense: 5′-GAAGCAGCTCTCATCCTACAA-3′ and #2 sense: 5′-CGTCTCTTCAAAGCCATGATA-3′.

Li Y., Zhang X., Liu N., Liu R., Zhang W., Chen L, & Chen Y. (2024). RNF166 promotes colorectal cancer progression by recognizing and destabilizing poly-ADP-ribosylated angiomotins. Cell Death & Disease, 15(3), 211.

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Investigating TNFAIP3-mediated NF-κB regulation

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Antibodies specific for TNFAIP3 N-terminus (sc-166692), TRAF6 (sc-8409, sc-7221), Actin (sc-1615), Ubc13 (sc-376470, sc-292618), TNFR1 (sc-8436), RIP (sc-7881, sc-133102), NEMO (sc-56919, sc-8330), Ub (sc-271289), Caspase-1 (sc-515 and sc-622), GFP (sc-8334), HA-probe (sc-7392), c-MYC (sc-40) from Santa Cruz; TNFAIP3 N-terminal specific (#5630), K63-linkage Polyubiquitin (#5621), Phospho-IKKα/β (#2697), IKKα (#11930), IκBα (#4814, #9242), Phospho-IκBα (#2859), NFκB P65 (#8242), TNFR1 (#3736), RIP (#3493), Phospho-p38 MAPK (#4511), Phospho-p44/42 MAPK (Erk1/2) (#4370), Phospho-SAPK/JNK (#4668), p44/42 MAPK (Erk1/2) (#4695), SAPK/JNK (#9252), p38 MAPK (#8690), HRP-linked anti-rabbit IgG (#7074), HRP-linked anti-mouse IgG (#7076) from Cell Signaling; IL-1 β (AF-201-NA) from R&D Systems; NLRP3 (ALX-804-819-C100) from Enzo Life Sciences. pRK5-HA-Ubiquitin-K63 (Addgene plasmid # 17606) was a gift from Ted Dawson⁴³. pEGFP-C1-TNFAIP3 (Addgene plasmid # 22141) was a gift from Yihong Ye ⁴⁴. Myc-DDK-tagged-human RIPK1 (RC216024), Myc-DDK-tagged-human NFKBIA (RC200711), untagged human TRAF6 (sc109845) were from Origene. 3xFlag-TNFAIP3 (Ex-K6040-M120) was from Genecopoeia. pEF-NEMO was a gift from Dr. Chi Ma⁴⁵. The GFP tagged or 3xFlag-TNFAIP3 mutant plasmids were constructed by PCR mutagenesis.

Zhou Q., Wang H., Schwartz D.M., Stoffels M., Park Y.H., Zhang Y., Yang D., Demirkaya E., Takeuchi M., Tsai W.L., Lyons J.J., Yu X., Ouyang C., Chen C., Chin D.T., Zaal K., Chandrasekharappa S.C., Hanson E.P., Yu Z., Mullikin J.C., Hasni S.A., Wertz I., Ombrello A.K., Stone D.L., Hoffmann P., Jones A., Barham B.K., Leavis H.L., van Royen-Kerkof A., Sibley C., Batu E.D., Gül A., Siegel R.M., Boehm M., Milner J.D., Ozen S., Gadina M., Chae J., Laxer R.M., Kastner D.L, & Aksentijevich I. (2015). Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early onset autoinflammatory syndrome. Nature genetics, 48(1), 67-73.

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Constructing IPMK, Ubiquitin, and TRAF6 Plasmids

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pCMV-GST full-length IPMK and fragments were constructed as described previously (42 (link)). pRK5-HA-Ubiquitin-K48 (#17605) and pRK5-HA-Ubiquitin-K63 (#17606) constructs were purchased from Addgene. pcDNA3.0 FLAG-TRAF6 and fragments were gifts from E.-K. Jo and S. Y. Lee. pCEP-HA-IRAK1 was a gift from Y.-J. Song.

Kim E., Beon J., Lee S., Park S.J., Ahn H., Kim M.G., Park J.E., Kim W., Yuk J.M., Kang S.J., Lee S.H., Jo E.K., Seong R.H, & Kim S. (2017). Inositol polyphosphate multikinase promotes Toll-like receptor–induced inflammation by stabilizing TRAF6. Science Advances, 3(4), e1602296.

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ITCH E3 Ligase Substrate and Regulation

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Full-length human ITCH cDNA was amplified from human Hela cDNA and cloned into the pcDNA3.1 (Xpress-tag, Invitrogen) and pET-32a(+) (Novagen) vectors. Ala substitution at S257 of ITCH and Arg substitution at K46 of H1.2 was performed using QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies). WT ITCH, S257A ITCH, WT H1.2, and K46R H1.2 cDNAs were cloned into pEGFP-C1 (Clontech), a gift from Dr. Yanzhong Yang (City of Hope). ITCH deletion mutants were generated by PCR and subcloned into Xpress-tag pcDNA3.1. ITCH shRNA and scrambled control shRNA were purchased from MISSION shRNA at Sigma-Aldrich. HEK293T or MDA-MB-231 cells were transfected using PolyJet DNA transfection Reagent (SignaGen). DDK-tagged H1.2, H1.3 and H1.4 plasmids were purchased from OriGene Technologies, Inc. pRK5-HA-ubiquitin-K63, pRK5-HA-ubiquitin-K48, and pRK5-HA-ubiquitin-K29 were purchased from Addgene.

Chang L., Shen L., Zhou H., Gao J., Pan H., Zheng L., Armstrong B., Peng Y., Peng G., Zhou B.P., Rosen S.T, & Shen B. (2018). ITCH nuclear translocation and H1.2 polyubiquitination negatively regulate the DNA damage response. Nucleic Acids Research, 47(2), 824-842.

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Plasmid Constructs for Gene Expression

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pTRE2-Bla-cRILP and pTRE2-Bla-ncRILP have been described^{27 (link)}. pFRT-TODest-FLAGHAhFMRPiso1 (Addgene plasmid 48690) was a gift from Thomas Tuschl^{28 (link)}. HA-FMR1 was generated by excising the gene from pFRT and inserting into pLVX-IRES-mCherry followed by site-directed mutagenesis (NEB) to remove the Flag tag. All sequences were confirmed by DNA sequencing analysis. The pLKO-based plasmids to express shRNAs targeting TRIM25 (TRCN0000272649) and TRC1 empty vector control (SHC001) were obtained from Sigma-Aldrich. pRK5-HA-Ubiquitin-K63 was a gift from Ted Dawson (Addgene plasmid # 17606)^{29 (link)}. 155_KRAS in pmiRGlo was a gift from Heidi Schwarzenbach (Addgene plasmid # 78132)^{30 (link)}.

King K.E., Ghosh P, & Wozniak A.L. (2023). TRIM25 dictates selective miRNA loading into extracellular vesicles during inflammation. Scientific Reports, 13, 22952.

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