Plasmids purchased from Addgene included: pLKO1-TRC (10878), pcDNA3-myr-HA-AKT1 (46969), pcDNA3-HA-AKT1 (73408), pcDNA3-HA-AKT1-K179M (73409), pcDNA3-HA-AKT1-1-149aa (73410), pcDNA3-HA-AKT1-120-433aa (73411), pRK5-HA-Ubiquitin-WT (17608), pRK5-HA-Ubiquitin-K29 (22903), and pRK5-HA-Ubiquitin-K29R (17602). Plasmids p3.3 empty vector, p3.3-Myc-Ubiquitin-WT, p3.3-Myc-Ubiquitin-K48, p3.3-Myc-Ubiquitin-K63, p3.3-flag-KLHL19, p3.3-flag-KLHL21, p3.3-flag-KLHL22, p3.3-flag-ZNRF1, p3.3-flag-ZNRF2, p3.3-flag-BACURD1, p3.3-flag-BACURD2, p3.3-flag-RNF152, p3.3-flag-RNF167, p3.3-flag-β-Trcp1, p3.3-flag-FBW7, p3.3-flag-HERC5, and p3.3-flag-Skp2 were provided by Jie Chen at Beijing University in China. pcDNA3 empty vector was purchased from Invitrogen. pMD.G and p8.74 were from PlasmidFactory. Human pITA-flag-CASTOR1 WT was cloned from 293T cells. Rat pITA-flag-CASTOR1 WT was previously described13 . The mutants of human pITA-flag-CASTOR1, including S14A, S14D, K61R, K96R, K213R, K61R/K96R, K61R/K213R, and K61R/K96R/K213R, were generated using a mutagenesis kit (NEB E0554) based on the manufacturer’s instructions. The primer sequences used for the cloning are listed in Table S1 and the sequences of all plasmids were confirmed by direct sequencing.
Prk5 ha ubiquitin k29
Manufactured by Addgene
PRK5-HA-Ubiquitin-K29 is a plasmid that contains the Ubiquitin gene with a K29 mutation and an HA tag. It is designed for the expression and study of ubiquitin proteins.
Automatically generated - may contain errors
Lab products found in correlation
Plko 1 trc, by Addgene (1 mentions)
Pcdna3 ha akt1, by Addgene (1 mentions)
Prk5 ha ubiquitin wt, by Addgene (1 mentions)
Pcdna3 myr ha akt1, by Addgene (1 mentions)
Polyjet dna transfection reagent, by SignaGen (1 mentions)
Prk5 ha ubiquitin k48, by Addgene (1 mentions)
Xpress tag, by Thermo Fisher Scientific (1 mentions)
Pegfp c1, by Takara Bio (1 mentions)
Prk5 ha ubiquitin k63, by Addgene (1 mentions)
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions)
2 protocols using prk5 ha ubiquitin k29
1
Plasmid Cloning and Mutagenesis Protocol
2
ITCH E3 Ligase Substrate and Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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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.
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