For transient transfection, subconfluent cells were transfected with 2 μg plasmid of pECE‐flag‐Sirt1 (Addgene, Cambridge, MA, USA), pECE empty vector (Addgene), HA‐HIF‐1α‐wt‐pcDNA3, HA‐HIF‐1‐K709R‐pcDNA3, pGL2‐hypoxia‐responsive element (HRE) plasmid (Addgene) using Trans IT 2020 (Mirus Bio LLC, Madison, WI) following the manufacturer's instructions. For luciferase assay, pGL2 luciferase reporter vector (Addgene) for HRE was cotransfected with Firefly and Gaussia luciferase control plasmid using a Dual Luciferase Assay kit (Thermo Scientific, Rockford, IL, USA). Luciferase activity was measured using a luminometer.
Pece flag sirt1
Manufactured by Addgene
Sourced in United States
PECE-flag-Sirt1 is a plasmid construct that expresses the SIRT1 protein with a FLAG epitope tag. SIRT1 is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that plays a role in various cellular processes, including metabolism, stress response, and aging. The FLAG tag allows for detection and purification of the SIRT1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Pece empty vector, by Addgene (2 mentions)
Dual luciferase assay kit, by Thermo Fisher Scientific (1 mentions)
Transit 2020, by Mirus Bio (1 mentions)
Gw6471, by Merck Group (1 mentions)
Nicotinamide, by Beyotime (1 mentions)
Compound c, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
N acetyl l cysteine nac, by Beyotime (1 mentions)
2 protocols using pece flag sirt1
1
Transient Transfection for HIF-1α Assay
2
Human Liver Cell Line L02 Response
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Human embryonic liver cell line L02 was purchased from China Center for Type Culture Collection (Wuhan, China). N-acetyl-L-cysteine (NAC) (Beyotime, Shanghai, China) (5 mmol/L)[22 (link)], nicotinamide (NAM) (Beyotime) (5 mmol/L)[23 (link)], GW6471 (Sigma-Aldrich) (3 μM)[24 (link)] or Compound C (Sigma-Aldrich) (10 μM)[25 (link)], which were dissolved in dimethyl sulfoxide (Sigma-Aldrich), were used to pretreat L02 cells for 1 h, followed by LPS (5 μg/mL)[26 (link)] treatment. Hypoxic conditions (1% O2) were obtained using humidified variable aerobic workstation InVivo2 400 (Ruskinn, Pencoed, United Kingdom)[27 (link)]. For transient transfection, cells were transfected with 2 μg plasmid of pECE-flag-Sirt1 (Addgene, Cambridge, MA, United States) and pECE empty vector (Addgene).
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