Protocol detail

ATF4-Luciferase Assay for ER Stress

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The experiment was performed as previously described (Wong et al., 2018 (link)). Briefly, HEK293T cells expressing an ATF4-luciferase reporter (Sidrauski et al., 2013 (link)) were seeded into 96-well plates and treated with 100 nM thapsigargin for 7 hr to induce ER stress. Cells were co-treated with 2BAct or ISRIB in dose response. Luminescence was measured using ONE-Glo Luciferase assay reagent (Promega) and a Molecular Devices SpectraMax i3x plate reader. Data were analyzed in Prism (GraphPad Software).

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Wong Y.L., LeBon L., Basso A.M., Kohlhaas K.L., Nikkel A.L., Robb H.M., Donnelly-Roberts D.L., Prakash J., Swensen A.M., Rubinstein N.D., Krishnan S., McAllister F.E., Haste N.V., O'Brien J.J., Roy M., Ireland A., Frost J.M., Shi L., Riedmaier S., Martin K., Dart M.J, & Sidrauski C. (2019). eIF2B activator prevents neurological defects caused by a chronic integrated stress response. eLife, 8, e42940.

Publication 2019

ONE-Glo Luciferase assay reagent SpectraMax i3x plate reader

Assay Atf4 Cells Devices Luciferase Luminescence Prism Promega Thapsigargin

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Variable analysis

independent variables

Dose of 2BAct
Dose of ISRIB

dependent variables

Luminescence (measured using ONE-Glo Luciferase assay reagent and a Molecular Devices SpectraMax i3x plate reader)

control variables

HEK293T cells expressing an ATF4-luciferase reporter
Treatment with 100 nM thapsigargin for 7 hr to induce ER stress

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