His ubiquitin

Manufactured by Addgene

His-Ubiquitin is a recombinant protein consisting of a histidine (His) tag fused to the ubiquitin protein. Ubiquitin is a small regulatory protein found in most tissues of eukaryotic organisms and plays a crucial role in protein degradation and other cellular processes. The His tag allows for easy purification of the ubiquitin protein using affinity chromatography methods.

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Lab products found in correlation

Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Ab92742, by Abcam (1 mentions) Dynabeads protein g 10004d, by Thermo Fisher Scientific (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Phospho 4ebp1, by Cell Signaling Technology (1 mentions) Gfp rab11 wt, by Addgene (1 mentions)

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Ubiquitin (3 citations)

3 protocols using his ubiquitin

Mammalian Expression Vectors for Nrf2 Study

Cited 1 time

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The mammalian expression vectors pcDNA3-EGFP-C4-Nrf2 (EGFP-Myc-WT-Nrf2) #21549, pGLUE-HA-FBXW11#36969, and His-Ubiquitin (#31815) were obtained from Addgene and pcDNA3.1 vector from Invitrogen. The triple serine–alanine mutant form of Nrf2 (EGFP-MycTM-Nrf2) was designed as previously described [65 (link)].

Petsouki E., Ender S., Sosa Cabrera S.N, & Heiss E.H. (2023). AMPK-Mediated Phosphorylation of Nrf2 at S374/S408/S433 Favors Its βTrCP2-Mediated Degradation in KEAP1-Deficient Cells. Antioxidants, 12(8), 1586.

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Investigating mTOR Pathway Regulation

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Everolimus (SML2282) was purchased from Sigma-Aldrich; Merck KGaA and dissolved in DMSO. Dynabeads Protein G (10004D) and TRIzol reagent were purchased from Invitrogen. Antibodies against OTUD5 (PA5-20611) and RNF186 (PA5-42330, PA5-57315) were purchased from Thermo Fisher Scientific. Antibodies against mTOR (2983), Phospho-mTOR (Ser2448) (5536,2976), Phospho-p70 S6 Kinase (Thr421/Ser424) (9204), Phospho-4E-BP1 (Thr37/46) (2855), Cleaved-PARP (5625), Cleaved-caspase3 (9664), Flag-tag (14793), HA-tag (3724), GST-tag (2624) and β-actin (3700) were purchased from Cell Signaling Technology. Antibodies against Ki-67 (ab92742) and Bax (ab32503) were purchased from Abcam. Antibody against sestrin2 (10795-1-AP) was purchased from Proteintech Group. pcDNA3-Flag-OTUD5, pcDNA3-HA-SESN1, pcDNA3-HA-SESN2, pcDNA3-HA-SESN3, pcDNA3-HA-RNF186 and pGEX-4T-1-GST-OTUD5 plasmids were constructed according to our standard protocols. The construct expressing His-ubiquitin was purchased from Addgene. pLKO-shOTUD5 (TRCN0000233196, TRCN0000143838) was purchased from Sigma-Aldrich.

Hou T., Dan W., Liu T., Liu B., Wei Y., Yue C., Que T., Ma B., Lei Y., Wang Z., Zeng J., Fan Y, & Li L. (2022). Deubiquitinase OTUD5 modulates mTORC1 signaling to promote bladder cancer progression. Cell Death & Disease, 13(9), 778.

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Plasmid Construction and Characterization

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Backbone of the plasmid pcDNA4-TO-RNF43-2xHA-2xFLAG (kindly gifted by Bon-Kyoung Koo together with pcDNA4-TO-RNF43Mut1-2xHA-2xFLAG (Koo et al., 2012) (link) Other plasmids used were described previously and included: myc-Vangl1, GFP-Vangl2, GFP-Vangl2ΔN, GFP-Vangl2ΔC, GFP-Vangl2ΔNΔC (Belotti et al., 2012) (link), pEGFP-C1-Rab5a (Chen et al., 2009) (link), GFP-rab11 WT (Addgene #12674), His-Ubiquitin (Tauriello et al., 2010) (link), pcDNA3-Flag-mDvl1 (Tauriello et al., 2010) (link), pCMV5-3xFlag Dvl2 (Addgene #24802), pCDNA3.1-Flag-hDvl3 (Angers et al., 2006) (link) Finally, PCR products were sequenced using the Illumina platform and compared with the reference sequence (Malcikova et al., 2015) (link). Sequencing results are presented in the Supplementary Table 1.

Radaszkiewicz T., Nosková M., Gömöryová K., Blanářová O.V., Radaszkiewicz K.A., Pícková M., Víchová R., Gybeľ T., Kaiser K., Demková L., Kučerová L., Potěšil D., Zdráhal Z., Souček K., & Bryja V. (2021). RNF43 inhibits WNT5A driven signaling and suppresses melanoma invasion.

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