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Pcdna3 myr ha akt1

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PcDNA3 Myr-HA Akt1 is a plasmid vector that contains the Akt1 gene with a myristoylation signal and HA tag. The vector is designed for the expression of the Akt1 protein in mammalian cells.

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Lab products found in correlation

Ha ubiquitin, by Addgene (2 mentions) Pcdna3 jnk1a1 apf dn jnk, by Addgene (2 mentions) Pmcs green renilla luc vector, by Thermo Fisher Scientific (2 mentions) Pcmv red firefly luc vector, by Thermo Fisher Scientific (2 mentions) Plko 1 trc, by Addgene (1 mentions) Pcdna3 ha akt1, by Addgene (1 mentions) Prk5 ha ubiquitin wt, by Addgene (1 mentions) Prk5 ha ubiquitin k29, by Addgene (1 mentions) Pcdna3 flag ha akt1, by Addgene (1 mentions) Pcw57.1 4ebp1 4xala, by Addgene (1 mentions) Smartpool, by Horizon Discovery (1 mentions) Pcdna3 ha gsk 3βs9a, by Addgene (1 mentions) Pcdna3 myr ha akt2, by Addgene (1 mentions)

Spelling variants of this product

PcDNA3.1 (119 citations) PcDNA3-HA-AKT1 (5 citations) Myr-Akt1 (4 citations) PcDNA3-HA (3 citations) 1036 pcDNA3 Myr HA Akt1 (3 citations)

7 protocols using pcdna3 myr ha akt1

1

Plasmid Cloning and Mutagenesis Protocol

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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.
Li T., Wang X., Ju E., da Silva S.R., Chen L., Zhang X., Wei S, & Gao S.J. (2021). RNF167 activates mTORC1 and promotes tumorigenesis by targeting CASTOR1 for ubiquitination and degradation. Nature Communications, 12, 1055.
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2

Plasmid Constructs for Diabetes Research

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pCMV-myc-MST1 and kinase-dead (MST1-K59; dnMST1) was kindly provided by Dr. Junichi Sadoshima and Dr. Yasuhiro Maejima (UMDNJ, New Jersey Medical School) 30 (link). Mouse pB.RSV.PDX1-GFP plasmid was kindly provided by Dr. Ingo Leibiger (Karolinska University, Stockholm). pcDNA3 Myr-HA Akt1, HA-Ubiquitin and pCDNA3 Jnk1a1 (apf)(dn-JNK) plasmids were obtained from Addgene (Cambridge, MA). Mouse PDX1 mutants (T11, T126, T152, T155, T214 and T231) in pCGIG5 vector were generated by site-directed mutagenesis as described previously 38 (link). All mutations were verified by sequencing. To make bacterial expression plasmids for PDX1 mutants, the complete mouse PDX1 CDS (wild type and mutants) has been amplified by PCR using a specific set of primers from pCGIG5 plasmids and cloned into a pGEX-6P-1 bacterial expression vector (kindly provided by Dr. Reinhard Walther, University of Greifswald). The rat insulin driven luciferase vector (RIP-Luc) was constructed by subcloning a 700bp fragment containing -660bp of the rat 2 insulin promoter (kindly provided by Dr. Rolf Zinkernagel, University of Zurich) into a pMCS-Green-Renilla-Luc vector (Thermo Scientific). pCMV-Red firefly Luc vector was obtained from Thermo Scientific.
Ardestani A., Paroni F., Azizi Z., Kaur S., Khobragade V., Yuan T., Frogne T., Tao W., Oberholzer J., Pattou F., Conte J.K, & Maedler K. (2014). MST1 is a novel regulator of apoptosis in pancreatic beta-cells. Nature medicine, 20(4), 385-397.
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3

Plasmid Expression Constructs for FOXO Signaling

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Expressing plasmids, including pcDNA3-Flag-FKHR(FOXO1)-Delta DB, pcDNA3-flag FKHRL1 (FOXO3a), pcDNA3-Flag-HA, pcDNA3-Flag-HA-Akt1, pcDNA3-Myr-HA-Akt1, and pcDNA3-T7-Akt1-T308A-S473A [34 (link)] were purchased from Addgene (Cambridge, MA, USA). The HBP1 expressing plasmid pEF-BOS-HA-HBP1, and FOXO1 expressing plasmids pcDNA3-A3-FOXO1-Flag pcDNA3-WT-FOXO1-Flag and pcDNA3-H215R-FOXO1-Flag were kindly provided by Dr. Amy S. Yee and Dr. Brian Schaffhausen (Tufts University, USA), respectively.
Chan C.Y., Huang S.Y., Sheu J.J., Roth M.M., Chou I.T., Lien C.H., Lee M.F, & Huang C.Y. (2017). Transcription factor HBP1 is a direct anti-cancer target of transcription factor FOXO1 in invasive oral cancer. Oncotarget, 8(9), 14537-14548.
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4

Generation of Myr-AKT and 4EBP1-4A Cell Lines

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MCF-7 cells overexpressing a myristoylated form of AKT (Myr-AKT) were generated by transfection with 1 µg of pcDNA3-Myr-HA-AKT1 (Addgene plasmid #9008). Transfection was aided by pre-incubation with lipofectamine 2000 and used according to the manufacturer’s instructions. Media was changed 6 hours after transfection, and cells were harvested after an additional 24 hours. MDA-MB-468 and MCF-7 cells expressing inducible 4EBP1 T37A/T46A/S65A/T70A (4EBP1–4A) were generated by lentiviral transduction with pCW57.1–4EBP1_4xAla (Addgene plasmid #38240)48 . Lentivirus were packaged in 293T cells, and viral supernatant was filtered with 0.45-µm PVDF filters and incubated with target cells for six hours. Cells were cultured in virus-free media for two days, and infected cells were selected using Puromycin (2 μg/ml) for 3 days.
Lee B.J., Boyer J.A., Burnett G.L., Thottumkara A.P., Tibrewal N., Wilson S.L., Hsieh T., Marquez A., Lorenzana E.G., Evans J.W., Hulea L., Kiss G., Liu H., Lee D., Larsson O., McLaughlan S., Topisirovic I., Wang Z., Wang Z., Zhao Y., Wildes D., Aggen J.B., Singh M., Gill A.L., Smith J.A, & Rosen N. (2021). Selective Inhibitors of mTORC1 Activate 4EBP1 and Suppress Tumor Growth. Nature chemical biology, 17(10), 1065-1074.
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5

Manipulating PI3K/Akt and FOXO3a Signaling

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pcDNA3Myr HA-Akt1 was from Dr William Sellers (Addgene plasmid #9008). pCMV-GFP was from Dr. Connie Cepko (Addgene plasmid #11153). HA-FOXO3a wild type (WT) (Addgene Plasmid #1787), HA-FOXO3a WT DBM (H212R) (Addgene Plasmid #8352) and HA-FOXO3a TM (Addgene Plasmid #1788) were gifts from Dr Michael Greenberg. pCMV-Ubc9 ( RC217884 ) was obtained from Origene. The SAE2-GFP gene was PCR-amplified from pCMV-SAE2-GFP and inserted into pLenti CMV/TO Puro DEST (670-1) to obtain a Tet-On-inducible SAE2-GFP expression plasmid. siRNAs against SAE2, UBC9 and Dicer were obtained from GE Dharmacon SMARTpool.
Li Y.J., Du L., Aldana-Masangkay G., Wang X., Urak R., Forman S.J., Rosen S.T, & Chen Y. (2018). Regulation of miR-34b/c-targeted gene expression program by SUMOylation. Nucleic Acids Research, 46(14), 7108-7123.
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6

Plasmid Constructs for Diabetes Research

Cited 1 time
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pCMV-myc-MST1 and kinase-dead (MST1-K59; dnMST1) was kindly provided by Dr. Junichi Sadoshima and Dr. Yasuhiro Maejima (UMDNJ, New Jersey Medical School) 30 (link). Mouse pB.RSV.PDX1-GFP plasmid was kindly provided by Dr. Ingo Leibiger (Karolinska University, Stockholm). pcDNA3 Myr-HA Akt1, HA-Ubiquitin and pCDNA3 Jnk1a1 (apf)(dn-JNK) plasmids were obtained from Addgene (Cambridge, MA). Mouse PDX1 mutants (T11, T126, T152, T155, T214 and T231) in pCGIG5 vector were generated by site-directed mutagenesis as described previously 38 (link). All mutations were verified by sequencing. To make bacterial expression plasmids for PDX1 mutants, the complete mouse PDX1 CDS (wild type and mutants) has been amplified by PCR using a specific set of primers from pCGIG5 plasmids and cloned into a pGEX-6P-1 bacterial expression vector (kindly provided by Dr. Reinhard Walther, University of Greifswald). The rat insulin driven luciferase vector (RIP-Luc) was constructed by subcloning a 700bp fragment containing -660bp of the rat 2 insulin promoter (kindly provided by Dr. Rolf Zinkernagel, University of Zurich) into a pMCS-Green-Renilla-Luc vector (Thermo Scientific). pCMV-Red firefly Luc vector was obtained from Thermo Scientific.
Ardestani A., Paroni F., Azizi Z., Kaur S., Khobragade V., Yuan T., Frogne T., Tao W., Oberholzer J., Pattou F., Conte J.K, & Maedler K. (2014). MST1 is a novel regulator of apoptosis in pancreatic beta-cells. Nature medicine, 20(4), 385-397.
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7

Generating Akt and GSK3β Mutant Constructs

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pcDNA3-Myr-HA-AKT1 (#9008), pcDNA3-Myr-HA-AKT2 (#9016), pcDNA3-Myr-HA-AKT3 (#9017) and pcDNA3-HA-GSK3β-S9A (#14754) were obtained from Addgene. We used overlap PCR method to produce AKT3 mutants AKT3-T305A, AKT3-S472A, AKT3-T305A/S472A (AKT3-AA), AKT3-K177M (AKT3-KD) and subcloned the WT AKT1-3 and AKT3 mutants into an AAV backbone that contained CBA promoter with Myr-3xHA tag at the N-terminus to get AAV-Myr-3HA-AKT1-3 and AKT3 mutants. We generated AAV-3HA-GSK3β-S9A similarly and AAV-Cre, AAV-S6K1-DN, AAV-S6K1-CA and AAV-4E-BP1-4A were described before (Yang et al., 2014 (link)).
Miao L., Yang L., Huang H., Liang F., Ling C, & Hu Y. (2016). mTORC1 is necessary but mTORC2 and GSK3β are inhibitory for AKT3-induced axon regeneration in the central nervous system. eLife, 5, e14908.
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