AMG-18

IRE 1 biochemical fluorescence quenching kinase assay: A truncated human IRE1α WT protein, containing the catalytic part (AA 469-977), was expressed in insect cells. The purified protein (1.2 μg/mL) was incubated with a fluorescently quenched IRE1α substrate (1.45 μg/mL; 5’ [6FAM] – GAG UCC GCA GCA CUC - [BHQ1] 3’) and a titration of inhibitor, starting at 50 μM (final DMSO concentration = 1%), for 40 min at room temperature in assay buffer. Reactions were carried out in a final volume of 20 μL in a 384 well PP plate (Greiner #784209). The reaction was stopped by addition of 15 μL 100 mM Tris-HCl pH9 and the fluorescence intensity of the IRE1α-substrate was measured using a fluorescent plate reader (Ex 485 nm / Em 535 nm, Perkin Elmer Envision).