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Pbabe puro ha pik3ca

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PBabe-puro-HA PIK3CA is a plasmid that contains the coding sequence for the catalytic subunit of the phosphoinositide 3-kinase (PI3K) enzyme, specifically the PIK3CA isoform. The plasmid includes an HA (hemagglutinin) tag for detection and a puromycin resistance marker for selection of transfected cells.

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Lab products found in correlation

Pbabe puro ha pik3ca e545k, by Addgene (1 mentions) Polybrene, by Santa Cruz Biotechnology (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Plenti pgk hygro dest, by Addgene (1 mentions) 293ft cell, by Thermo Fisher Scientific (1 mentions) Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions) Sanger sequencing, by Genewiz (1 mentions) In fusion cloning, by Takara Bio (1 mentions) Quikchange 2 xl, by Agilent Technologies (1 mentions)

4 protocols using pbabe puro ha pik3ca

1

Validating Cell Lines and Analyzing PIK3CA Mutations

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The human SYO-1 (Kawai et al., 2004 (link)) and Yamato-SS (Naka et al., 2010 (link)) cell lines were maintained in DMEM with 10% FBS, and validated every 3 mo by expression of and dependence (siRNA) on SS18-SSX2 or 1, respectively. Transfection with pBabe-puro-HA, pBabe-puro-HA PIK3CA, or pBabe-puro-HA PIK3CA (H1047R; Addgene) used Lipofectamine 3000 (Invitrogen). LY294002 (5 µM) treatments began 24 h after transfection and lasted for 48 h.
Barrott J.J., Kafchinski L.A., Jin H., Potter J.W., Kannan S.D., Kennedy R., Mosbruger T., Wang W.L., Tsai J.W., Araujo D.M., Liu T., Capecchi M.R., Lazar A.J, & Jones K.B. (2016). Modeling synovial sarcoma metastasis in the mouse: PI3′-lipid signaling and inflammation. The Journal of Experimental Medicine, 213(13), 2989-3005.
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2

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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3

Lentiviral Vectors for PIK3CA Expression

Cited 2 times
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Third generation lentiviral gateway destination vector (pLenti-PGK-Hygro-DEST, #19066, a gift from Eric Campeau and Paul Kaufman), pENTR4 vector (pENTR4-no-ccDB, #17424, a gift from Eric Campeau and Paul Kaufman), hemagglutinin (HA)-tagged wild-type (WT) PIK3CA (PIK3CAWT, pBabe-puro-HAPIK3CA, #12522, a gift from Jean Zhao), and HA-tagged GFP (GFP, pDEST-Flag-HA-GFP, #22612, a gift from Wade Harper) plasmids were purchased from Addgene (Cambridge, MA) [36 (link)–38 (link)]. Wild-type PIK3CA (PIK3CAWT) and GFP were excised and inserted into pENTR4 vector by ligation. PIK3CAmut (R88Q, C90Y, E542K, E545K, M1043V, H1047R) were generated by point mutagenesis of PIK3CAWT using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA) per manufacturer’s instructions. GFP, PIK3CAWT, and PIK3CAmut were transferred from pENTR4 to pLenti-PGK-Hygro-DEST vectors by recombination as described [36 (link)]. All mutations were confirmed by Sanger sequencing (Genewiz, South Plainfield, NJ). Lentiviral particles encoding GFP, PIK3CAWT, or individual PIK3CAmut were generated in 293FT cells (Invitrogen, Grand Island, NY) per manufacturer’s instructions.
McNeill R.S., Stroobant E.E., Smithberger E., Canoutas D.A., Butler M.K., Shelton A.K., Patel S.D., Limas J.C., Skinner K.R., Bash R.E., Schmid R.S, & Miller C.R. (2018). PIK3CA missense mutations promote glioblastoma pathogenesis, but do not enhance targeted PI3K inhibition. PLoS ONE, 13(7), e0200014.
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4

Cloning and Mutagenesis of PIK3CA

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pBabe puro HA-PIK3CA was purchased from Addgene (plasmid #12522). This construct has an artifactual amino acid change in its coding sequence (I143V) and site-directed mutagenesis (Quikchange II XL, Agilent) was performed to convert it back to WT with the two primers in the Table above. The coding sequence of PIK3CA was then cloned PIK3CA into the pcDNA3.4 vector through in-fusion cloning (Clontech) as an untagged protein or with an N-terminal polyhistidine tag. Sequencing analysis was performed with Snapgene.
Lin T.Y., Ramsamooj S., Perrier T., Liberatore K., Lantier L., Vasan N., Karukurichi K., Hwang S.K., Kesicki E.A., Kastenhuber E.R., Wiederhold T., Yaron T.M., Huntsman E.M., Zhu M., Ma Y., Paddock M.N., Zhang G., Hopkins B.D., McGuinness O., Schwartz R.E., Ersoy B.A., Cantley L.C., Johnson J.L, & Goncalves M.D. (2023). Epinephrine inhibits PI3Kα via the Hippo kinases. Cell reports, 42(12), 113535.
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