FIGURE 1.

Recombinant expression and activity of the purified cytosolic human IRE1α kinase endoribonuclease. A, expression strategy for a cytosolic fragment (amino acids 462–977) of human IRE1α (hIRE1α-cyto) using a baculovirus/insect cell system. hIRE1α-cyto was liberated from GST using PreScission protease. B, SDS-polyacrylamide gel electrophoresis analysis of GST-fused and PreScission-liberated hIRE1α-cyto. C, schematic of the mini-XBP-1 stem-loop used as the hIRE1α-cyto substrate for HTS and IC₅₀ analysis; the Cy5 fluorophore was linked to the 5′ end and black hole quencher 2 (BHQ2) was linked to the 3′ end. D, time course analysis of mini-XBP-1 substrate cleavage (100 nm starting concentration) by the indicated concentrations of hIRE1α-cyto.