Pcdna3 gfp pten

Manufactured by Addgene

The pcDNA3 GFP PTEN is a plasmid vector that contains the coding sequence for the green fluorescent protein (GFP) and the tumor suppressor gene PTEN. This vector can be used for the expression and study of the PTEN protein in various cell types.

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Lab products found in correlation

Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions) Ha pkb t308d s473d pcdna3, by Addgene (1 mentions) Transit lt1 transfection reagent, by Mirus Bio (1 mentions) Rat tail collagen, by BD (1 mentions) Tgf β3, by Thermo Fisher Scientific (1 mentions) Anti mouse igg hrp, by GE Healthcare (1 mentions) Goat anti rabbit igg hrp, by Thermo Fisher Scientific (1 mentions) Smad2 3, by Cell Signaling Technology (1 mentions) P akt ser473, by Cell Signaling Technology (1 mentions) Pcdna3 gfp, by Addgene (1 mentions) Antibody against β actin clone ac 15, by Merck Group (1 mentions) Recombinant human tgf β1, by Thermo Fisher Scientific (1 mentions) P smad3, by Cell Signaling Technology (1 mentions) Matrigel, by BD (1 mentions) Transwell insert, by BD (1 mentions) Risc free control sirna, by Horizon Discovery (1 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions) Opti mem medium, by Thermo Fisher Scientific (1 mentions) Sirna, by Santa Cruz Biotechnology (1 mentions) Pcdna3 egfp, by Addgene (1 mentions)

4 protocols using pcdna3 gfp pten

Modulating Akt Signaling in Melanoma Cells

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HA-tagged pcDNA-AKT-AAA (pcDNA3 HA PKB AAA, kinase dead, Addgene) and HA-tagged pcDNA-AKT-DD (HA PKB T308D S473D pcDNA3, constitutively active, Addgene) (Scheid et al., 2002 (link)) were transfected into A375 cells using TransIT®-LT1 Transfection Reagent (Mirus Bio) according the manufacturer's instructions. GFP-tagged pcDNA-PTEN (pcDNA3 GFP PTEN, Addgene) (Vazquez et al., 2001 (link)) was transfected into SK-MEL-24 cells using Lipofectamine 3000 Reagent (Invitrogen) according to the manufacturer's instructions. Cells were seeded for cell proliferation assay 24 hours after transfection. Cells were harvested for Western blot 48 hours after transfection.

Lin R., Xia S., Shan C., Chen D., Liu Y., Gao X., Wang M., Kang H.B., Pan Y., Liu S., Chung Y.R., Abdel-Wahab O., Merghoub T., Rossi M., Kudchadkar R.R., Lawson D.H., Khuri F.R., Lonial S, & Chen J. (2018). Dietary supplement chondroitin-4-sulfate exhibits oncogene-specific pro-tumor effects on BRAF V600E melanoma cells. Molecular cell, 69(6), 923-937.e8.

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TGF-β Signaling Pathway Regulation

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Recombinant human TGF-β1 and TGF-β3 were purchased from PeproTech (Rocky Hill, NJ). Mammalian expression vectors pcDNA3 GFP PTEN (plasmid # 10759), and pcDNA3 GFP (plasmid #20738) were obtained from Addgene (Cambridge, MA). Human PTEN siRNA (sc-29459) and control non-silencing siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Dallas, TX). Matrigel, rat tail collagen and transwell inserts were purchased from BD Biosciences (Bedford, MA). DAPI (4′, 6-Diamidine-2-phenylindole dihdochloride) was purchased from Roche Diagnostics, (Indiana, IN). The antibodies against PTEN, pAKT^Ser473, AKT (pan), pSmad2, pSmad3, and Smad2/3 were purchased from Cell Signaling Technology (Danvers, MA). Antibody against β-Actin (clone AC-15) was purchased from Sigma-Aldrich (St. Louis, MO). Goat anti-rabbit IgG HRP was obtained from Life Technologies (Grand Island, NY). Anti-mouse IgG HRP was obtained from GE Healthcare (Piscataway, NJ).

Kimbrough-Allah M.N., Millena A.C, & Khan S.A. (2018). Differential Role of PTEN in Transforming Growth Factor β (TGF-β) Effects on Proliferation and Migration in Prostate Cancer Cells. The Prostate, 78(5), 377-389.

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Plasmid and siRNA Transfection Protocols

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The plasmids pCMV-HA-ISG15, pSG5-UBE1L, pCMV2-UBCH8, and pcDNA4-USP18 were described before [16 (link), 19 (link)]. Full-length PTEN cloned into a MYC-tagged expression vector [10 (link)] was obtained from Dr. Li Ma (MD Anderson Cancer Center). The pcDNA3-GFP-PTEN and the pCMV-GFP-USP18 plasmids were purchased (Addgene and GeneCopoeia). Deletions in PTEN were constructed (GENEWIZ). DNA sequence analysis confirmed the desired species were engineered. Respective vector controls were purchased. RISC-free control siRNA and two siRNAs independently targeting USP18 were purchased (GE Dharmacon). These sequences were: murine USP18 siRNA 1 (5′-CGTTGTTTGTCCAGCACGA-3′) and murine USP18 siRNA 2 (5′-AGGAACTCGAGGACGGAAA-3′). Plasmids and siRNAs were transfected into cells using Lipofectamine 2000 reagent (Invitrogen) and Opti-MEM medium (Gibco Thermo Scientific).

Mustachio L.M., Kawakami M., Lu Y., Rodriguez-Canales J., Mino B., Behrens C., Wistuba I., Bota-Rabassedas N., Yu J., Lee J.J., Roszik J., Zheng L., Liu X., Freemantle S.J, & Dmitrovsky E. (2016). The ISG15-specific protease USP18 regulates stability of PTEN. Oncotarget, 8(1), 3-14.

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Modulating Rho GTPases in MCF-7 Cells

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MCF-7 cells were cultured for 18 hrs before transient transfection with each plasmid using GenJet™ Reagent II for MCF-7 (Signagen, Ijamsville, MD) according to the manufacturer's instructions. The plasmid expression was confirmed through changes in cell shape, and immunoblot or fluorescence analyses. The following plasmids (Addgene, Cambridge, MA) were used: pCDNA3-EGFP (Addgene ID 13031), pCDNA3-EGFP-RhoA-Q63L (constitutively active RhoA, Addgene ID 12968), pCDNA3-EGFP-RhoA-T19N (dominant negative, Addgene ID 12967), pCDNA3-EGFP-Rac1-Q61L (constitutively active Rac1, Addgene ID 12981), pCDNA3-EGFP-Rac1-T17N (dominant negative, Addgene ID 12982), and pCDNA3-GFP-PTEN (wild type, Addgene ID 10759). For the RNA interference (RNAi) experiments, MCF-7 cells were seeded for 18 hrs and transfected with siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) using a siRNA transfection reagent (Santa Cruz Biotechnology, Santa Cruz, CA) according to manufacturer's instructions. The siRNA for targeting ROCK-1, ROCK-2, and PTEN were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA).

Yang S, & Kim H.M. (2014). ROCK Inhibition Activates MCF-7 Cells. PLoS ONE, 9(2), e88489.

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