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Prk5 ha gst ragc 75l

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PRK5-HA GST RagC 75L is a lab equipment product. It serves as a core function, but a detailed description while maintaining an unbiased and factual approach is not available.

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Lab products found in correlation

Prk5 ha gst raga 66l, by Addgene (2 mentions) Pegfp n1 tfeb, by Addgene (2 mentions) Myc mtor, by Addgene (1 mentions) Trpml1 ha, by Addgene (1 mentions) Pcdna3 flag rheb n153t, by Addgene (1 mentions) In fusion kit, by Takara Bio (1 mentions) Egfp rab7a q67l, by Addgene (1 mentions) Mucolipin1 pegfp c3, by Addgene (1 mentions) Plenti cmv mcs gfp sv puro, by Addgene (1 mentions) Pspax2, by Addgene (1 mentions) Pmd2 g, by Addgene (1 mentions) Pljc5 tmem192 3xha, by Addgene (1 mentions) Lr clonase, by Thermo Fisher Scientific (1 mentions) Bp clonase, by Thermo Fisher Scientific (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Fugene 6 reagent, by Promega (1 mentions)

4 protocols using prk5 ha gst ragc 75l

1

Generation of Recombinant Protein Expression Constructs

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Myc-mTOR (Addgene plasmid # 1861), pRK5-HA GST RagA 66L (Addgene plasmid # 19300), pRK5-HA GST RagC 75L (Addgene plasmid # 19305) and HA GST PreScission p70 S6K1 (Addgene plasmid # 15511) were gifts from David Sabatini. pcDNA3-FLAG-Rheb-N153T (Addgene plasmid # 19997) was a gift from Fuyuhiko Tamanoi. pcDNA-CaM was a gift from David Yue. TRPML1-HA (Addgene plasmid # 18825) was a gift from Craig Montell. EGFP-Rab7A Q67L (Addgene plasmid # 28049) and EGFP-Rab7A T22N (Addgene plasmid # 28048) were gifts from Qing Zhong.
FLAG-tagged RhebN153T was amplified by PCR and cloned into the EcoRI site of pLVX-AcGFP-N1 vector. GST-tagged 4EBP1 was amplified by PCR and cloned into a pDEST15-based vector. FLAG-tagged CaM was amplified by PCR and cloned into a pGEX6-based vector. TRPML1 was amplified by PCR and cloned into a pEGFP-based vector. All ligations were performed with Infusion Kit (Clontech Laboratories, Inc.) according to the manufacture’s instruction. After sequence verification, these plasmids were used, as described below, in transient cDNA transfections, bacterial protein expression or to produce the lentiviruses needed to generate cell lines stably expressing the proteins.
Li R.J., Xu J., Fu C., Zhang J., Zheng Y.G., Jia H, & Liu J.O. (2016). Regulation of mTORC1 by lysosomal calcium and calmodulin. eLife, 5, e19360.
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2

Generating EGFP-MCOLN1 Lentiviral Construct

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To obtain EGFP-MCOLN1 construct for lentiviral transduction, human MCOLN1 was amplified by PCR using the following oligonucleotides with overhangs (underlined): 5′-GACACCGACTCTAGAATGGTGAGCAAGGGCGAGGAGC-3′ (forward) and 5′-AACTAGTCCGGATCCTCAATTCACCAGCAGCGAATGC-3′ (reverse) from the mucolipin1-pEGFP C3 (plasmid #62960; Addgene) construct and subcloned into XbaI and BamHI restriction sites of the pLenti-CMV-MCS-GFP-SV-puro (plasmid #73582; Addgene) vector using the sequence- and ligation-independent cloning (SLIC) method as described elsewhere (Jeong et al, 2012 (link)). The mucolipin1-pEGFP C3 (plasmid #62960; Addgene) construct was a gift from Paul Luzio (Pryor et al, 2006 (link)). pRK5-HA GST RagC WT (plasmid #19304; Addgene) and pRK5-HA GST RagC 75L (plasmid #19305; Addgene) were a gift from David Sabatini (Sancak et al, 2008 (link)). psPAX2 (plasmid #12260; Addgene) and pMD2.G (plasmid #12259; Addgene) lentiviral packaging plasmids were a gift from Didier Trono.
Wróbel M., Cendrowski J., Szymańska E., Grębowicz-Maciukiewicz M., Budick-Harmelin N., Macias M., Szybińska A., Mazur M., Kolmus K., Goryca K., Dąbrowska M., Paziewska A., Mikula M, & Miączyńska M. (2022). ESCRT-I fuels lysosomal degradation to restrict TFEB/TFE3 signaling via the Rag-mTORC1 pathway. Life Science Alliance, 5(7), e202101239.
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3

Cloning and expression of human FNIP1

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The cDNA encoding human FNIP1 (Uniprot Q8TF40) was generated by reverse transcription polymerase chain reaction (RT-PCR) from RNA obtained from IMR90 cells. Fusion tag FLAG-CHERRY was added to the N-terminal end of FNIP1 and subcloned into pDONR221 with BP Clonase (Invitrogen). The cDNA for human FLCN was obtained from Invitrogen (IOH12359). Mammalian expression vectors were generated, by recombining ENTR clones into DEST vectors using LR Clonase (Invitrogen). Destination vectors include: pLentiCMV/TO (Addgene no. 17293), pcDNA3 N-term FLAG DEST, pcDNA3 N-Term MYC DEST, and pQCXIB CMV/TO (Addgene no. 17400). Site-directed mutagenesis for FNIP1 was performed using QuikChange II XL (Stratagene) according to the manufacturer’s instructions. Untagged human AMPKα1 cDNA was cloned into pLentiCMV/TO puro Gateway destination vector. Other plasmids used include pLJC5-Tmem192–2xFlag (41 (link)) (Addgene no. 102929), pLJC5-Tmem192–3xHA (41 (link)) (Addgene no. 102930), pEGFP-N1-TFEB (5 (link)) (Addgene no. 38119), pRK5-HA GST RagC 120L (Addgene no. 19306), and pRK5-HA GST RagC 75L (Addgene no. 19305).
Britton R.A, & Grossman A.D. (1999). Synthetic Lethal Phenotypes Caused by Mutations Affecting Chromosome Partitioning in Bacillus subtilis. Journal of Bacteriology, 181(18), 5860-5864.
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4

Plasmid Transfection Optimization for HeLa, HEK293T Cells

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The following plasmids were used in HeLa-TFEB-GFP cells: pRK5 HA- RAGA (#99710), pRK5-HA GST RAGA 66L (#19300), pRK5 HA- RAGC (#99718), and pRK5-HA GST RAGC 75L (#19305), were purchased from Addgene. The following plasmids were used in HeLa cells: TFEB-GFP wild type, TFEB-GFP S142A, TFEB-GFP S211A, TFEB-GFP S142A/S211A developed by Dr. Shawn Ferguson. The following plasmids were used in HEK293T cells: pEGFP-N1-TFEB (Addgene #38119), pCMV-Tag3B vector plasmid (Agilent) and pCMV-Tag3B expressing myc-FLCN plasmid57 (link). All plasmids were expressed using Fugene6 reagent (Promega) for 48 h in immunofluorescence experiments and using Lipofectamine 3000 (Invitrogen) for 6 h in immunoblotting experiments.
Alesi N., Akl E.W., Khabibullin D., Liu H.J., Nidhiry A.S., Garner E.R., Filippakis H., Lam H.C., Shi W., Viswanathan S.R., Morroni M., Ferguson S.M, & Henske E.P. (2021). TSC2 regulates lysosome biogenesis via a non-canonical RAGC and TFEB-dependent mechanism. Nature Communications, 12, 4245.
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