Flag sirt1

Manufactured by Addgene

Sourced in United States

Flag-SIRT1 is a tagged version of the SIRT1 protein. SIRT1 is a member of the sirtuin family of proteins involved in various cellular processes. The Flag tag allows for the detection and purification of the SIRT1 protein.

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Lab products found in correlation

Flag sirt1 h363y, by Addgene (2 mentions) Sirt1 sirna, by Santa Cruz Biotechnology (2 mentions) Ha akt dn k179 m, by Addgene (1 mentions) Myr akt, by Addgene (1 mentions) Pcdna3.1 gfp, by Addgene (1 mentions) Flag foxo3a, by Addgene (1 mentions) Ampk sirna, by Santa Cruz Biotechnology (1 mentions) Consirna, by Santa Cruz Biotechnology (1 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)

4 protocols using flag sirt1

FoxO3a Regulation in Mammalian Cells

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Mammalian expression vectors, pcDNA3.1-GFP, Flag-SIRT1, Flag-FoxO3a, WT-Akt, Myr-Akt, HA-Akt DN (K179M), and SIRT6 were purchased from Addgene. The shAkt and shFoxO3a vectors were described previously 8 (link), 9 (link). An empty vector was used as control. The shRNA reagents for SIRT1 and SIRT6 were purchased from Dharmacon.
The constitutively active FoxO3a (Ad-Tm-FoxO3a) is mutated at three phosphorylation sites, so that it cannot be phosphorylated. The dominant-negative FoxO3a (Ad-DN-FoxO3a) lacks the C-terminus transactivation domain 31 (link). For adenoviral expression, Ad-Tm-FoxO3a and DN-FoxO3a were cloned into Kpn I-BamH I of pShuttle-CMV-EGFP, followed by linearizing and co-transforming into DH5α with the adenoviral virus pAdeno (Invitrogen). The reconstituted adenoviral DNA containing FoxO3a cDNA was then transfected into 293 cells.

Zhang Y., Nie L., Xu K., Fu Y., Zhong J., Gu K, & Zhang L. (2019). SIRT6, a novel direct transcriptional target of FoxO3a, mediates colon cancer therapy. Theranostics, 9(8), 2380-2394.

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SIRT1 Overexpression in HK2 Cells

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HK2 cells were transfected with SIRT1, SIRT1^H363Y, or control plasmids to mimic the overexpression of SIRT1, the inactivated form of SIRT1 using a transient transfection technical method. Twenty-four h after transfection, cells were washed and processed for immunoblotting and other studies described above. Two plasmids, Flag-SIRT1 and Flag-SIRT1^H363Y were designed by Addgene Corporation¹^,² .

Xu S., Zeng Z., Zhao M., Huang Q., Gao Y., Dai X., Lu J., Huang W, & Zhao K. (2019). Evidence for SIRT1 Mediated HMGB1 Release From Kidney Cells in the Early Stages of Hemorrhagic Shock. Frontiers in Physiology, 10, 854.

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SIRT1 and AMPK Regulation in VSMC

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VSMC was transfected with control siRNA or siRNA against SIRT1 and AMPK by using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Con siRNA, SIRT1 siRNA, and AMPK siRNA were purchased from Santa Cruz (USA). Cells then were resuspended in complete DMEM, incubated for 24–48 hr, and used for further experiments. Flag-SIRT1 was obtained from Addgene plasmid repository (Addgene plasmid #1791; Greenberg ME, Harvard Medical School, Boston, MA, USA). For transfection, VSMC was seeded on 6-well plates and when 80% confluent, transfected in Opti-MEM/Lipofectamine 2000 Reagent containing SIRT1 plasmid DNA.

Sung J.Y., Kim S.G., Kim J.R, & Choi H.C. (2020). Prednisolone suppresses adriamycin-induced vascular smooth muscle cell senescence and inflammatory response via the SIRT1-AMPK signaling pathway. PLoS ONE, 15(9), e0239976.

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SIRT1 Expression Vector Transfection

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The human SIRT1 expression vector Flag-SIRT1 and Flag-SIRT1 H363Y (deacetylase domain mutation) were purchased from Addgene (Cambridge, MA, USA). Transient transfections with 1μg plasmid DNA were performed with Lipofectamine 2000 (Invitrogen) at 1:3 ratio for 24-48 h. SIRT1 siRNA (Santa Cruz Biotechnology, USA) were used at 20 nM/2.5 × 10 5 cells with Lipofectamine 2000 (Invitrogen) for 48 h [21] .

Ray U., Roy S.S, & Chowdhury S.R. (2017). Lysophosphatidic Acid Promotes Epithelial to Mesenchymal Transition in Ovarian Cancer Cells by Repressing SIRT1. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 41(2).

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