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 empty vector
Manufactured by Addgene
Sourced in United States
The PECE empty vector is a plasmid that can be used as a backbone for cloning and expression in mammalian cells. It provides a basic framework for gene insertion and expression without any pre-existing genes or regulatory elements.
Automatically generated - may contain errors
Lab products found in correlation
Pece flag sirt1, 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 empty vector
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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