Transient Transfection and Reporter Assay

Reporter plasmids pTATA-LUC, pPSA5.8-LUC^{15 (link)}, pFKBP51(-3)-LUC (FKBP51-1)^{16 (link)}, pFKBP51(12)-LUC (FKBP51-2)^{17 (link)} (Promega, US), and pCMVβ encoding β-galactosidase are purified from E. coli JM109 strain cultures using QIAGEN Plasmid Maxi Kit (Qiagen, Germany). Transfection of LNCaP and VCaP were carried out using jetPEITM transfection reagent according to manufacturer’s protocols (Polyplus-Transfection, France). Reporter gene assay was performed using TransDetect® luciferase reporter assay kit (TransGen, China) and Luciferase assay system (Promega, USA). The β-galactosidase-normalized LUC activities of each groups were documented. Experiments were performed at least three times.

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Quan Z., Li T., Xia Y., Liu J., Du Z., Luo C., He Y, & Wu X. (2020). PLCɛ maintains the functionality of AR signaling in prostate cancer via an autophagy-dependent mechanism. Cell Death & Disease, 11(8), 716.

Publication 2020

QIAGEN Plasmid Maxi Kit jetPEITM transfection reagent Luciferase assay system

Assay E coli Fkbp51 Luciferase Plasmid Promega Reporter gene Strain Transfection Vcap β galactosidase

Corresponding Organization :

Other organizations : First Affiliated Hospital of Chongqing Medical University, Chongqing Medical University, Second Affiliated Hospital of Chongqing Medical University, Dalian Medical University

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Variable analysis

independent variables

Reporter plasmids (pTATA-LUC, pPSA5.8-LUC, pFKBP51(-3)-LUC, pFKBP51(12)-LUC)
Cell lines (LNCaP, VCaP)

dependent variables

Luciferase (LUC) activities (normalized to β-galactosidase)

control variables

Transfection reagent (jetPEITM)
Luciferase reporter assay kit (TransDetect® luciferase reporter assay kit)
Luciferase assay system (Promega, USA)
Plasmid purification method (QIAGEN Plasmid Maxi Kit)
E. coli strain (JM109)

controls

PCMVβ encoding β-galactosidase (positive control)

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