Prk5 myc raptor

Manufactured by Addgene

PRK5-myc-Raptor is a plasmid that expresses the human raptor protein fused with a myc tag. Raptor is a key regulatory subunit of the mTOR complex 1 (mTORC1). The plasmid can be used for the study of mTORC1 signaling pathways.

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Lab products found in correlation

Prk5 myc rictor, by Addgene (2 mentions) Pp242, by Selleck Chemicals (1 mentions) Pepstatin a, by Cayman Chemical (1 mentions) Prk5 ha raptor, by Addgene (1 mentions)

3 protocols using prk5 myc raptor

Evaluating Cell Line Responses to Pharmacological Agents

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ATCC supplied all human cancer cells and normal mouse kidney (TCMK-1) cells (Manassas, VA), and Lonza supplied normal human mesangial cells (Basel, Switzerland). All cell lines tested negative for mycoplasma contamination. The lines were authenticated by standard morphologic examination using microscopy. Cells were cultured in DMEM containing 10% FBS and 100 μg/mL gentamycin. R&D system supplied z-VAD-fmk and TNF-α (Minneapolis, MN), and Biomol supplied curcumin (Plymouth Meeting, PA). LKT Labs (St. Paul, MN) and Selleckchem (Huston, TX) supplied Kahweol and PP242, respectively. Calbiochem supplied Quercetin, Luteolin, 2-aminoethosxydiphenyl borate (2-APB), and EGTA-AM (San Diego, CA). Enzo Life Sciences (Plymouth Meeting, PA) and Cayman Chemical (Ann Arbor, MI) supplied pepstatin A and E64D and, respectively. Sigma Chemical Co. supplied other chemicals (St. Louis, MO). We provided information for used antibodies in Supplementary Table 1. Human Akt1 cDNA (Upstate Biotechnology, Lake Placid, NY) was cloned into pcDNA3.1-Myc/His vector. pRK5 vector was purchased from Clontech Laboratories, Inc. (Mountain View, CA). pRK5-myc-Raptor (Addgene plasmid # 1859) and pRK5-myc-Rictor (Addgene plasmid # 1859) were a gift from David Sabatini [52 (link)]. Santa Cruz Biotechnology supplied the siRNA (Santa Cruz, CA).

Seo S.U., Woo S.M., Lee H.S., Kim S.H., Min K.J, & Kwon T.K. (2018). mTORC1/2 inhibitor and curcumin induce apoptosis through lysosomal membrane permeabilization-mediated autophagy. Oncogene, 37(38), 5205-5220.

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Overexpression and Mutagenesis of Raptor and TSC2

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We used pRK5-HA-Raptor (Addgene 8513) and pRK5-myc-Raptor (Addgene 1859) plasmids to overexpress the HA-and myctagged forms of human Raptor. The pcDNA3-FLAG-TSC2 plasmid (Addgene 14129) mammalian expression vector was used to overexpress the WT form of human TSC2. We used this plasmid as a template to create, by PCR driven mutagenesis, the pcDNA3-FLAG-TSC2 S1217A , pcDNA3-FLAG-TSC2 S1452A and pcDNA3-FLAG-TSC2 S1217A, S1452A phosphorylation mutant constructs, where the indicated serines of TSC2 were mutated to alanines. The appropriate mutations were verified by sequencing. Rb and Metap2 were PCR-amplified from HEK293T cDNA and cloned into pcDNA3-based vectors. Myc-S6K was generated by subcloning the human S6K1 coding sequence from the Addgene plasmid 100509 into a pcDNA3-based vector containing a myc-tag.

Romero-Pozuelo J., Figlia G., Kaya O., Martin-Villalba A, & Teleman A.A. (2020). Cdk4 and Cdk6 Couple the Cell-Cycle Machinery to Cell Growth via mTORC1. Cell reports, 31(2).

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Characterizing mTOR Pathway Regulators

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Cells were transfected with 10 μg of DNA using 30 μg of linear polyethylenimine. The following vectors were used: pcDNA3.1 Flag-eYFP, pLPC Flag-KDM4A, pCMV-Sport6-Flag-mTOR, pMSCV HA-DEPTOR, pDEST myc-KDM4A wild type or mutants (H188A and D939R), pcDNA3 Myc-β-TrCP1 (Addgene plasmid #20718; gift from Dr Yue Xiong) and pcDNA3 HA-Ub. The following plasmids, pRK5 Myc-mTOR (Addgene plasmid #1861), pRK5 Myc-Raptor (Addgene plasmid #1859), pRK5 Myc-Rictor (Addgene plasmid #11367), pRK5 Flag-DEPTOR (Addgene plasmid #21334), pRK5 Flag-DEPTOR DEP domains (Addgene plasmid #21700) and pRK5 Flag-DEPTOR PDZ domain (Addgene plasmid #21701) were gifts from Dr David Sabatini.

Carbonneau M., M. Gagné L., Lalonde M.E., Germain M.A., Motorina A., Guiot M.C., Secco B., Vincent E.E., Tumber A., Hulea L., Bergeman J., Oppermann U., Jones R.G., Laplante M., Topisirovic I., Petrecca K., Huot M.É, & Mallette F.A. (2016). The oncometabolite 2-hydroxyglutarate activates the mTOR signalling pathway. Nature Communications, 7, 12700.

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