Figure 1.

ATG4B activity is promoted by reductant and reversibly inhibited by oxidant. (A-B) Activity of recombinant His-ATG4B was monitored by quantitative cleaved GST product in a dose-dependent (A) and time-dependent manner (B). (C) His-ATG4B (0.015 µg) was pre-treated with different concentration of DTT for 10 min, after which ATG4B was incubated with LC3-GST (5 µg) in reaction buffer at 37℃ for 30 min. 0ʹ represents LC3-GST only without ATG4B or any other treatments. (D) His-ATG4B (0.025 µg) was incubated in the presence of indicated H₂O₂ for 30 min, after which LC3-GST (5 µg) was added and reaction proceeded at 37℃ for 30 min. (E) His-ATG4B (0.015 µg) was laid and exposed to air on ice for up to 3 h, then ATG4B cleaved LC3-GST (5 µg) in the absence or presence of DTT (1 mM). (F) His-ATG4B (0.015 µg) was pretreated with 0.2 mM of DTT for 10 min (lane 3); then, H₂O₂ (1 mM) was added for 30 min (lane 4); finally, oxidized ATG4B was treated with 2 mM of DTT for another 30 min (lane 5). After each specific procedure except blank control (lane 1), ATG4B was incubated with LC3-GST to assess activity. All the reaction mixtures were subject to SDS-PAGE, followed by Coomassie Brilliant Blue (CBB) staining. Data are presented as mean ± SEM from 4 independent assays. *** P < 0.001.