Fig. 2

Ebselen is a highly active and selectivity inhibitor of ATG4B. A, B Cell lysates (1.5 μg) from ATG4B-Flag 293 T were incubated with different compounds (10 μM) for 30 min at 37 °C, then recombinant LC3B-GST (3 μg) were added up to 20 μl and inhibitions were detected by SDS-PAGE and CBB staining (A). B Statistical chart of gel results of three repeated experiments. Ebselen was abbreviated as Ebs, the same below. CON as control without lysate, NC as negative control without compound. C, D Inhibition rate of Ebselen for ATG4A and ATG4B determined by FRET assay (C) and validated by SDS-PAGE (D). CON as control without enzyme, NC as negative control without compound. E Target selectivity of Ebselen (10 μM) was tested against caspase-3 (CASP3). Z-VAD-FMK, 50 μM. CON as control without enzyme, NC as negative control without compound. F Target selectivity of Ebselen (10 μM) was tested against caspase proteases (CASP1, 2, 4, 6, 8, and 9). CON as control without Ebselen. G Structure list of analogues based on benzo[d] [1,2]selenazol-3-one scaffold. H The inhibitory effect of analogues (10 μM) was detected by SDS-PAGE and CBB staining