Pbabe puro ha pik3ca e545k

Manufactured by Addgene

Sourced in United States

The PBabe-puro-HA-PIK3CA-E545K is a plasmid that contains the gene encoding the PIK3CA-E545K mutant protein with an HA tag. It is intended for use in research applications.

Automatically generated - may contain errors

Lab products found in correlation

Pbabe b raf v600e, by Addgene (2 mentions) Pt2 bh transposon vectors, by Addgene (2 mentions) Pbabe puro ha pik3ca, by Addgene (1 mentions) Polybrene, by Santa Cruz Biotechnology (1 mentions) Pegfp c2, by Takara Bio (1 mentions) Pcdna3.1 v5 his topo, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Qiaquick gel extraction kit, by Qiagen (1 mentions) Fugene hd, by Promega (1 mentions) Plasmid midi kit, by Qiagen (1 mentions) Puromycin, by Thermo Fisher Scientific (1 mentions) Pbabe puro ha pik3ca h1047r, by Addgene (1 mentions) Pbabe puro, by Addgene (1 mentions) Zeocin, by Thermo Fisher Scientific (1 mentions) Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions) Sicontrol, by Horizon Discovery (1 mentions) Plenti6 h2b mcherry, by Addgene (1 mentions) On targetplus, by Horizon Discovery (1 mentions) Pspax2, by Addgene (1 mentions) Pmd2 g, by Addgene (1 mentions)

8 protocols using pbabe puro ha pik3ca e545k

Retroviral Transduction of PIK3CA Variants

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pBabe-puro-HA-PIK3CA and pBabe-puro-HA-PIK3CA-E545K were purchased from Addgene (#12522 and #12525 respectively). Retrovirus were produced by transfecting 293T cells with the expression plasmids and the two packaging vectors VSVG and pHIT60, using PEI (3μg per ug of DNA). Viral supernatants were collected after 72h, filtered though a 0.45μm filter and used to infect target cells in addition with Polybrene (8μg/ml, SantaCruz). After 24h, cells were washed and put under antibiotic selection for 96h (Puromycin 1μg/ml).

Romano G., Chen P.L., Song P., McQuade J.L., Liang R.J., Liu M., Roh W., Duose D.Y., Carapeto F.C., Li J., Teh J.L., Aplin A.E., Chen M., Zhang J., Lazar A.J., Davies M.A., Futreal P.A., Amaria R.N., Zhang D.Y., Wargo J.A, & Kwong L.N. (2018). A pre-existing rare PIK3CAE545K subpopulation confers clinical resistance to MEK plus CDK4/6 inhibition in NRAS melanoma and is dependent on S6K1 signaling. Cancer discovery, 8(5), 556-567.

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Generation of Activated TAZ, BRAF, and PI3K Constructs

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Transposons encoding the constitutively activated forms of TAZ (pT3/EF5a TAZS89A) was a kind gift from Dr. Xin Chen at the University of California, San Francisco. The plasmids pT2/EGFP and pPGK-SB13 were described previously [27 (link)]. Open reading frames (ORFs) encoding the activated forms of BRAF (BRAF^V600E) and PIK3CA (PI3K^E545K) were polymerase chain reaction (PCR)-amplified from pBabe-B-RAF-V600E (#17,544; Addgene, Watertown, MA, USA) and pBabe-puro-HA-PIK3CA-E545K plasmids (#12,525; Addgene), respectively. These amplified PCR products were cloned into pT2/BH transposon vectors (#26,556; Addgene) to generate the plasmids, pT2/BRAF^V600E, and pT2/PI3K^E545K, respectively.

Moon H., Park H., Chae M.J., Choi H.J., Kim D.Y, & Ro S.W. (2022). Activated TAZ induces liver cancer in collaboration with EGFR/HER2 signaling pathways. BMC Cancer, 22, 423.

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Plasmid constructs for MKRN1 and PI3K/AKT proteins

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The MKRN1 open-reading frame (ORF)-containing plasmids pcDNA3.1-MKRN1 WT/H307E, pcDNA3-HA-MKRN1 WT/H307E, pcDNA3-FLAG-MKRN1 WT/H307E and pGEX-4T-1-GST-MKRN1 WT/H307E were described previously23 (link). MKRN1 S109A/S109D plasmids were generated by site-directed mutagenesis and subcloned into a pcDNA3-FLAG vector. The full-length PTEN ORF was kindly provided by Han-Woong Lee (Yonsei University) and subcloned into pcDNA3-HA or pcDNA3-FLAG. The AKT1 ORF-containing plasmids pLNCX-Myr-HA-Akt1-WT and K179M were purchased from Addgene (Cambridge, MA, USA), and the AKT1 ORF was subcloned into pcDNA3.1. The PIK3CA ORF-containing plasmids pBabe-puro-HA-PIK3CA E545K and H1047R were purchased from Addgene. pcDNA3-His-Ub was kindly provided by D.P. Lane38 (link). pHM6-HA-Ub has been described previously39 (link). pEGFP-C2 (Clontech, San Diego, CA) was used as a transfection control.

Lee M.S., Jeong M.H., Lee H.W., Han H.J., Ko A., Hewitt S.M., Kim J.H., Chun K.H., Chung J.Y., Lee C., Cho H, & Song J. (2015). PI3K/AKT activation induces PTEN ubiquitination and destabilization accelerating tumourigenesis. Nature Communications, 6, 7769.

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Cloning and Transfection of PIK3CA Variants

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The HA PIK3CA E545K fragment from pBABE puro HA PIK3CA E545K (Addgene, Cambridge, MA, USA) (plasmid number 12525) (Zhao et al, 2005 (link)) was cloned into pcDNA3.1/V5-His-TOPO (Life Technologies) and verified by sequencing. QiaQuick Gel extraction kit and Qiagen Plasmid Midi kit (Qiagen, Hilden, Germany) were used as per the manufacturer's protocol. pCMV2-Tag 2A PIK3CA-WT was purchased from Addgene (plasmid number 16643) (Samuels et al, 2004 (link)) and verified by sequencing. pcDNA3.1/V5-His-TOPO was used as a control. Cells were transfected using FuGENE HD (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. siRNA and DNA co-transfection was performed by using Lipofectamine 2000 (Life Technologies) as per the manufacturer's protocol. All assays were conducted 48 or 72 h after transfection.

Sathe A., Guerth F., Cronauer M.V., Heck M.M., Thalgott M., Gschwend J.E., Retz M, & Nawroth R. (2014). Mutant PIK3CA controls DUSP1-dependent ERK 1/2 activity to confer response to AKT target therapy. British Journal of Cancer, 111(11), 2103-2113.

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Retroviral Transduction of MYOD1 and PIK3CA Mutants

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For retrovirus production, the pcx4 [28 (link)]and pBabe vectors system were used. Human MYOD1 and MYC cDNA plasmids were purchased from Sino Biological Inc and subcloned into pcx4bleo. MYOD1 Leu122Arg mutant was generated using the QuikChange II site-directed mutagenesis kit (Agilent Technologies, La Jolla, CA, USA) and subcloned into pcx4bleo. pBabe-puro (Addgene plasmid 1764), pBabe puro HA PIK3CA H1047R (Addgene plasmid 12524) and pBabe puro HA PIK3CA E545K (Addgene plasmid 12525) were obtained from Addgene (Cambridge, MA, USA). Retroviruses were obtained by using 293T cells as packaging cells, and were infected into C2C12 and BJ cell lines and selected with 500 μg/ml Zeocin or 2 μg/ml puromycin (Invitrogen, Carlsbad, CA, USA).

Kohsaka S., Shukla N., Ameur N., Ito T., Ng C.K., Wang L., Lim D., Marchetti A., Viale A., Pirun M., Socci N.D., Qin L.X., Sciot R., Bridge J., Singer S., Meyers P., Wexler L.H., Barr F.G., Dogan S., Fletcher J.A., Reis-Filho J.S, & Ladanyi M. (2014). A recurrent neomorphic mutation in MYOD1 defines a clinically aggressive subset of embryonal rhabdomyosarcoma associated with PI3K/AKT pathway mutations. Nature genetics, 46(6), 595-600.

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Knockdown and overexpression of TRIP13 and related genes

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pLKO.1 containing scrambled control shRNA was obtained from Addgene (#1864), pLKO.1 vector sets with shRNA against TRIP13 were obtained from Sigma‐Aldrich. The following clones resulted in strong knockdown of Trip13 and were selected for further experiments: clones TRCN0000022063 and TRCN0000022059 for human TRIP13, TRCN0000319690 for mouse TRIP13. pLenti6‐H2B‐mCherry was a gift from Torsten Wittmann (Addgene plasmid # 89766). pHR_dSV40‐Aurora A–GFP was a gift from Ron Vale (Addgene plasmid # 67924). pEGFP‐C1‐ADRP was a gift from Elina Ikonen (Addgene plasmid # 87161). pLenti6‐ADRP was generated by subcloning. pBabe puro HA PIK3CA E545K was a gift from Jean Zhao (Addgene plasmid # 12525). psPAX2 (Addgene plasmid # 12260) and pMD2.G (Addgene plasmid # 12259), both were a gift from Didier Trono.
ON‐TARGETplus siRNAs were from Dharmacon: siControl (D‐001810‐01), siTrip13 (L‐016262‐00), siAurora A (L‐003545‐01), siACLY (L‐004915‐00), siACC (L‐004551‐00), siAGPAT2 (L‐003811‐00), siCD36 (L‐010206‐00), siINSR (L‐003014‐00), Mad2 (L‐003271‐00), siPten (L‐003023‐00), siMad2 (L‐003271‐00), siPlin1 (L‐019595‐01), siPlin2 (L‐019204‐01), siPlin3 (L‐015979‐00), siPlin4 (L‐184319‐00), siPlin5 (L‐033568‐01).

Rios Garcia M., Meissburger B., Chan J., de Guia R.M., Mattijssen F., Roessler S., Birkenfeld A.L., Raschzok N., Riols F., Tokarz J., Giroud M., Gil Lozano M., Hartleben G., Nawroth P., Haid M., López M., Herzig S, & Berriel Diaz M. (2022). Trip13 Depletion in Liver Cancer Induces a Lipogenic Response Contributing to Plin2‐Dependent Mitotic Cell Death. Advanced Science, 9(29), 2104291.

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Ectopic Expression of Key Signaling Proteins

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Plasmid 9018: 1271 pBabe puroL Myr HA Akt2, Plasmid 12523: pBabe puro Myr HA PIK3CA, Plasmid 12525: pBabe puro HA PIK3CA E545K, and Plasmid 15269: pBabe-Puro-BRAF-V600E were purchased from Addgene (Cambridge, MA). Transfection of HUVECs was done in six-well plates. Transfection reagents were mixed, vortexed, and incubated at room temperature for 30 min. Reagents (per six-well) were 95 μl of Opti-MEM Reduced Serum Medium with GlutaMAX (#51985-034; Invitrogen, Waltham, MA), 2 μl of siRNA (60 nM final concentration), and 4 μl of HiPerFect Transfection Reagent (#301705; Qiagen, Venlo, Netherlands). Cells were split and seeded at a density of 30–40% confluency in 650 μl of complete EGM-2 medium. Transfection reagents were added dropwise, and the cells were incubated overnight. The next day, 700 μl of EGM-2 was added (transfection reagents were not removed), and cells were incubated for additional 48 h. Transfected HUVECs were then split and seeded out in coculture with PaSMCs, as described in the following section.

Hellesøy M, & Lorens J.B. (2015). Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis. Molecular Biology of the Cell, 26(14), 2698-2711.

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Activated YAP, TAZ, BRAF, and PIK3CA Transposon Vectors

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Transposons encoding the constitutively activated forms of YAP (pT3/EF5a YAPS127A) and TAZ (pT3/EF5a TAZS89A) were kind gifts from Dr. Xin Chen at the University of California, San Francisco. The plasmids pT2/EGFP and pPGK-SB13 were described previously (Moon et al. 2017 ). Open reading frames (ORFs) encoding the activated forms of BRAF (BRAF V600E ) and PIK3CA (PI3K E545K ) were polymerase chain reaction (PCR)-ampli ed from pBabe-B-RAF-V600E (#17544; Addgene, Watertown, MA, USA) and pBabe-puro-HA-PIK3CA-E545K plasmids (#12525; Addgene), respectively. These ampli ed PCR products were cloned into pT2/BH transposon vectors (#26556; Addgene) to generate the plasmids, pT2/ BRAF V600E , and pT2/ PI3K E545K , respectively.

Moon H., Choi H.J., Kim D.Y., & Ro S.W. (2021). Activated TAZ Induces Liver Cancer in Collaboration with EGFR/HER2 Signaling Pathways.

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