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Development of a Novel Reporter System to Monitor ER stress
Author(s) -
Moon Ja Young,
Back Sung Hoon,
Han Jaeseok,
Kaufman Randal
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.834.15
Subject(s) - luciferase , fusion protein , reporter gene , microbiology and biotechnology , recombinant dna , hela , western blot , chemistry , transcription factor , cell culture , biology , transfection , gene expression , biochemistry , gene , genetics
A new reporter system to monitor ER stress by measuring ATF6a activation was developed. For this, four recombinant reporter vectors were constructed by using two promoters, pCMV or pTK, followed by coding sequences of Gal4 DNA‐binding domain (BD) andVP16 activation domain (AD) and C‐terminal part of human ATF6a (aa362–670 or aa374–670). As expected, the GAL4 DB‐VP16 AD‐hATF6a fusion protein expressed from the recombinant expression vectors was localized in the ER. Western blot analysis confirmed that both GAL4 DB‐VP16 AD‐hATF6a (aa362–670 or aa374–670) fusion proteins were cleaved to GAL4‐VP16 fragment and hATF6a (aa362–670 or aa374–670) proteins by ER stress. The PathDetect HeLa Luciferase Reporter (HLR) cell line stably established with pCMV‐GAL4‐VP16‐hATF6a(aa374–670) expression vector showed 7‐fold induction of dual luciferase activity by DTT treatment. In conclusion, the HLR‐GVATF6 cell line could be used as a novel reporter system to monitor ER stress by measuring luciferase activity induced by GAL4‐DB and VP16‐AD chimeric transcription factor during ER stress.