Figure 3.

ATG4B forms intramolecular disulfide bonds upon oxidation. (A) Reversible and irreversible cysteine modifications. (B) HEK293 cells transiently transfected with Flag-ATG4B or not were resolved in reduced (containing DTT) or non-reduced (DTT free) loading buffer and analyzed with anti-FLAG antibody. The relative level of Flag-ATG4B expression compared to endogenous control was determined with anti-ATG4B antibody. (C) Cell lysates (25 µg) from (B) were incubated with DTT ranging from 0.1 mM to 5 mM at 4℃ for 15 min, after which non-reduced SDS-PAGE was performed with anti-FLAG antibody. (D-E) Cell lysates (25 µg) from HEK293 cells transiently transfected with Flag-ATG4B (D) and HEK293 cells (E) were subject to non-reduced immunoblot assay after these procedures: treatment with 0.2 mM of DTT at 4℃ for 15 min (lane 1 in D, lane 2 in E); pretreatment with 0.2 mM of DTT followed by 1 mM of H₂O₂ for 30 min (lane 2 in D, lane 3 in E); 2 mM of DTT was added to mixture as detailed in lane 2 (lane 3 in D, lane 4 in E); no treatment (lane 4 in D, lane 1 in E). Reduced form of ATG4B (R) and oxidized form of ATG4B (O) were labeled. (F) HEK293 cells transiently transfected with plasmids of Flag-ATG4B or 13 single-site mutants (cysteine to serine) of ATG4B were lysed and subject to non-reduced immunoblot with anti-FLAG antibody after 24 h adherent growth. (G) Lysates from HEK293 cells overexpressing Flag-ATG4B or single-site mutant Flag-ATG4B^C292S, Flag-ATG4B^C361S or double-site mutant Flag-ATG4B^C292,361S (Flag-ATG4B^2CS) were incubated with H₂O₂ (1 mM) for 30 min. (H) ATG4B KO HeLa cells stably transfected with Flag-ATG4B or Flag-ATG4B^C292,361S were treated with H₂O₂ (1 mM) at 37℃ for 1 h, or cell lysates extracted from ATG4B KO HeLa cells stably transfected with Flag-ATG4B or Flag-ATG4B^C292,2361S were exposed to H₂O₂ (1 mM) for 30 min. Samples were subject to non-reduced SDS-PAGE and analyzed with anti-FLAG antibody.