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Prk5 ha ub k63

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PRK5-HA-Ub-K63 is a plasmid construct that expresses a fusion protein consisting of a hemagglutinin (HA) tag, ubiquitin with a lysine 63 (K63) mutation, and the protein kinase D5 (PRK5) domain. This plasmid can be used for research purposes, but a more detailed description of its core function or intended use cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

Prk5 ha ub k48, by Addgene (6 mentions) Prk5 ha ub, by Addgene (3 mentions) Prk5 ha ub wt, by Addgene (3 mentions) β actin antibody, by Santa Cruz Biotechnology (2 mentions) Anti xpress r91025, by Thermo Fisher Scientific (2 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions) Sc 8008, by Santa Cruz Biotechnology (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Pcdna3 ha ub, by Addgene (1 mentions) Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions) Ltx reagent, by Thermo Fisher Scientific (1 mentions) Prk5 ha ub k0, by Addgene (1 mentions) Plus reagent, by Thermo Fisher Scientific (1 mentions) Anti myc, by Santa Cruz Biotechnology (1 mentions)

Close spelling variants of this product

HA-Ub (16 citations) PRK5-HA-Ub (5 citations) HA-K63 (4 citations) HA-Ub-K63 (3 citations) Ub-K63 (2 citations)

6 protocols using prk5 ha ub k63

1

Transfection of SphK1 and RAGE Mutants

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The vector (pcDNA3 plasmid), Flag-tagged human wild-type SphK1 (SphKWT) and the dominant-negative SphK1 (SphKG82D) were kindly provided by Dr. Pu Xia (Australia) [36 (link)]. Vector pEGFP, human full-length wild-type pEGFP-RAGE and human cytoplasmic-deleted mutant-type pEGFP-RAGEΔcyto were constructed and supplied by Genechem. PcDNA3-HA-Ub, pcDNA3-HA-Ub K0, vector pRK5, pRK5-HA-Ub, pRK5-HA-Ub K48, and pRK5-HA-Ub K63 were purchased from Addgene (http://www.addgene.org/). GMCs were plated in 35 mm culture plates 24 h prior to transfection. Then, the cells were transfected with 2 μg indicated plasmid using Lipofectamine® LTX & Plus Reagent. GMCs were incubated for 72 h to harvest, and western blot assay or SphK activity detection was performed.
Chen C., Gong W., Li C., Xiong F., Wang S., Huang J., Wang Y., Chen Z., Chen Q., Liu P., Lan T, & Huang H. (2017). Sphingosine kinase 1 mediates AGEs-induced fibronectin upregulation in diabetic nephropathy. Oncotarget, 8(45), 78660-78676.
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2

Regulation of RSK2 and TRAF6 in Cellular Signaling

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Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were from Life Technologies, Inc. (Grand Island, NY). Antibodies against phosphor-RSK2 (#11989), phosphor-Ikkα/β (#2697), phosphor-IKBα (#2859), phosphor-JNKs (#4668), phosphor-p38 (#4511) and COX2 (#12282) were from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies against TRAF6 (sc-8409), Ub (sc-166553), and NFκB p65 (sc-8008) were from Santa Cruz Biotechnology, Inc. β-actin antibody was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Anti-Xpress (R91025) was from Thermo Fisher Scientific Corporation (Carlsbad, CA). Active RSK2 and anti-Flag (F3165) were from Upstate Biotechnology, Inc. (Charlottesville, VA). The pRK5-HA-Ub-Wt, pRK5-HA-Ub-K48 and pRK5-HA-Ub-K63 plasmids were purchased from Addgene (Cambridge, MA). The pCDNA3-Flag-TRAF6 plasmid was kindly provided by Dr. Peter ten Dijke (23 (link)), and the pCDNA4-Xpress-RSK2 plasmid was described previously (7 (link)).
Yao K., Lee S.Y., Peng C., Lim D.Y., Yamamoto H., Ryu J., Lim T.G., Chen H., Jin G., Zhao Z., Han Y., Ma W.Y., Bode A.M, & Dong Z. (2018). RSK2 is required for TRAF6 phosphorylation-mediated colon inflammation. Oncogene, 37(26), 3501-3513.
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3

Ubiquitin Chain Formation Regulation

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The plasmid pcDNA-5’ flag was a kind gift from Dr. Meng (Dalian Medical University). pRK5-HA-Ub and the mutant derivatives pRK5-HA-Ub-K48 and pRK5–HA-Ub-K63 were obtained from Addgene (plasmids #17608, #17605 and #17606) [19 (link)]. Attachment of ubiquitin (Ub) modifiers is a reversible post-translational modification that regulates the fate and function of proteins. The ubiquitin molecule contains a total of seven lysine residues at positions 6, 11, 27, 29, 33, 48 (K48), and 63 (K63). These lysine residues potentially mediate ubiquitin chain elongation. The two most common types of polyubiquitin chains are linked through ubiquitin lysine 48 (K48) and lysine 63 (K63).
Yu L., Zhang X., Wu T., Wang Y., Meng J., Liu Q., Niu X, & Wu Y. (2017). The papain-like protease of avian infectious bronchitis virus has deubiquitinating activity. Archives of Virology, 162(7), 1943-1950.
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4

Plasmid and siRNA transfection in HepG2 cells

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Flag-tagged PAQR3 plasmids and HA-tagged DDB2 plasmids were obtained from Vigene Bioscences (Jinan, China). pRK5-HA-Ub (RRID: Addgene_17608), pRK5-HA-Ub-K0 (RRID: Addgene_17603), pRK5-HA-Ub-K48 (RRID: Addgene_17605), and pRK5-HA-Ub-K63 (RRID: Addgene_17606) were obtained from Addgene (Watertown, MA, USA). Plasmids were handled according to the manufacturer's instructions with LTX reagent and PLUS reagent (ThermoFisher Scientific, Rockford, IL, USA). HepG2 cells were cultured 24 h prior to transfection, and then 2 μg of plasmid was transfected into cells with incubation for 48 h for further treatment.
The siRNA oligonucleotides of PAQR3 and DDB2 were purchased from GenePharma (Shuzhou, China), and the most effective sequences are listed in Supporting Information Table S3. Transfection of siRNA was performed according to the manufacturer's instructions with Lipofectamine RNAiMAX reagent (ThermoFisher Scientific). Transfected cells were incubated for 48 h for further treatment.
Xiao H., Sun X., Lin Z., Yang Y., Zhang M., Xu Z., Liu P., Liu Z, & Huang H. (2022). Gentiopicroside targets PAQR3 to activate the PI3K/AKT signaling pathway and ameliorate disordered glucose and lipid metabolism. Acta Pharmaceutica Sinica. B, 12(6), 2887-2904.
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5

Protein Overexpression and Co-Immunoprecipitation

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Calcium phosphate was used to transfect HEK293T cells with expression vectors for mouse RNF168 (WT and C21S mutant), HA-tagged WT or mutated mouse Rnf168, HA-RNF8, Flag-TOP2α, Flag-TOP2α−ϰR, HA-USP10 and WT or mutated HA-Ub (Addgene: pRK5-HA-Ub-WT (ID 17608), pRK5-HA-Ub-K48 [ID 17605), pRK5-HA-Ub-K63 (ID 17606)). 48 h post-transfection, cells were lysed in the modified RIPA buffer described earlier. IP was performed using anti-Flag, anti-HA (1:1,000; Santa Cruz, 080211) or anti-Myc (1:1,000; 9E10 Santa Cruz, sc40) antibodies and Protein A-Sepharose.
Guturi K.K., Bohgaki M., Bohgaki T., Srikumar T., Ng D., Kumareswaran R., El Ghamrasni S., Jeon J., Patel P., Eldin M.S., Bristow R., Cheung P., Stewart G.S., Raught B., Hakem A, & Hakem R. (2016). RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation. Nature Communications, 7, 12638.
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6

Regulation of RSK2 and TRAF6 in Cellular Signaling

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Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were from Life Technologies, Inc. (Grand Island, NY). Antibodies against phosphor-RSK2 (#11989), phosphor-Ikkα/β (#2697), phosphor-IKBα (#2859), phosphor-JNKs (#4668), phosphor-p38 (#4511) and COX2 (#12282) were from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies against TRAF6 (sc-8409), Ub (sc-166553), and NFκB p65 (sc-8008) were from Santa Cruz Biotechnology, Inc. β-actin antibody was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Anti-Xpress (R91025) was from Thermo Fisher Scientific Corporation (Carlsbad, CA). Active RSK2 and anti-Flag (F3165) were from Upstate Biotechnology, Inc. (Charlottesville, VA). The pRK5-HA-Ub-Wt, pRK5-HA-Ub-K48 and pRK5-HA-Ub-K63 plasmids were purchased from Addgene (Cambridge, MA). The pCDNA3-Flag-TRAF6 plasmid was kindly provided by Dr. Peter ten Dijke (23 (link)), and the pCDNA4-Xpress-RSK2 plasmid was described previously (7 (link)).
Yao K., Lee S.Y., Peng C., Lim D.Y., Yamamoto H., Ryu J., Lim T.G., Chen H., Jin G., Zhao Z., Han Y., Ma W.Y., Bode A.M, & Dong Z. (2018). RSK2 is required for TRAF6 phosphorylation-mediated colon inflammation. Oncogene, 37(26), 3501-3513.
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