Inhibition
of IRE1α endoribonuclease
activity was measured using a FRET de-repression assay monitoring
cleavage of a 29-nucleotide stem-loop RNA containing the XBP1 cleavage
site sequence and labeled with a fluorescence emitter (fluorescein
amidite (FAM)) and a fluorescence quencher (Black Hole Quencher (BHQ))
at the 5′ and 3′ ends, respectively (Figure S2 ), as described previously.30 (link),31 (link) Briefly, varying volumes of compound in DMSO or DMSO alone were
added to a low-volume 384-well plate (3676, Corning) to give final
concentrations ranging from 100 μM to 0.313 nM using an Echo
acoustic liquid dispenser (Labcyte, CA). Nonphosphorylated IRE1α
G547-L977 was added to a final concentration of 200 nM. After incubating
for 30 min at 30 °C, hairpin RNA XBP1 substrate mimic labeled
with fluorescein and Black Hole Quencher (5′ FAM-GAACAAGAUAUCCGCA-GCAUAUACAGUUC-3′
BHQ, Eurofins MWG Operon, Germany) was added to 100 nM final concentration.
After incubation for a further 15 min at 30 °C, the fluorescein
fluorescence was measured on an EnVision multimode plate reader (PerkinElmer
Life Sciences). Fluorescence in the presence of compound was expressed
relative to that of DMSO alone (no compound).
of IRE1α endoribonuclease
activity was measured using a FRET de-repression assay monitoring
cleavage of a 29-nucleotide stem-loop RNA containing the XBP1 cleavage
site sequence and labeled with a fluorescence emitter (fluorescein
amidite (FAM)) and a fluorescence quencher (Black Hole Quencher (BHQ))
at the 5′ and 3′ ends, respectively (
added to a low-volume 384-well plate (3676, Corning) to give final
concentrations ranging from 100 μM to 0.313 nM using an Echo
acoustic liquid dispenser (Labcyte, CA). Nonphosphorylated IRE1α
G547-L977 was added to a final concentration of 200 nM. After incubating
for 30 min at 30 °C, hairpin RNA XBP1 substrate mimic labeled
with fluorescein and Black Hole Quencher (5′ FAM-GAACAAGAUAUCCGCA-GCAUAUACAGUUC-3′
BHQ, Eurofins MWG Operon, Germany) was added to 100 nM final concentration.
After incubation for a further 15 min at 30 °C, the fluorescein
fluorescence was measured on an EnVision multimode plate reader (PerkinElmer
Life Sciences). Fluorescence in the presence of compound was expressed
relative to that of DMSO alone (no compound).
