Hypoxia-Induced HIF-1α Activity Assay

For HIF-1α luciferase activity assays, cells with ectopic Myc-Parkin expression or endogenous Parkin knockdown and their controls cells were transfected with the HIF-1α luciferase reporter vector for 12 h^{36 (link), 61 (link)}. Cells were then treated with or without hypoxia (1% O₂) for 16 h before being harvested for assays. Luciferase reporter assays were performed using the dual luciferase assay kit (Promega). The pRL-null vector expressing renilla luciferase (Promega) was used as an internal control to normalize the transfection efficiency.

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Liu J., Zhang C., Zhao Y., Yue X., Wu H., Huang S., Chen J., Tomsky K., Xie H., Khella C.A., Gatza M.L., Xia D., Gao J., White E., Haffty B.G., Hu W, & Feng Z. (2017). Parkin targets HIF-1α for ubiquitination and degradation to inhibit breast tumor progression. Nature Communications, 8, 1823.

Publication 2017

dual luciferase assay kit pRL-null vector expressing renilla luciferase

Assay Cells Ectopic expression Hypoxia Luciferase Parkin Promega Renilla luciferase Transfection Vector

Corresponding Organization :

Other organizations : Rutgers, The State University of New Jersey, First Affiliated Hospital Zhejiang University, Zhejiang University, Wenzhou Medical University

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Variable analysis

independent variables

Ectopic Myc-Parkin expression
Endogenous Parkin knockdown

dependent variables

HIF-1α luciferase activity

control variables

Normoxia (21% O2) conditions
Transfection efficiency (normalized by pRL-null vector expressing renilla luciferase)

controls

Positive control: Cells with ectopic Myc-Parkin expression or endogenous Parkin knockdown
Negative control: Control cells

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