Skip to main content
. 2020 Jan 3;16(10):1838–1850. doi: 10.1080/15548627.2019.1709763

Figure 4.

Figure 4.

ATG4B forms disulfide-linked intermolecular oligomers in response to oxidation. (A) HEK293 cells stably expressing Flag-ATG4B were incubated with increasing concentration of H2O2 for 1 h, then were lysed and modified with 10 mM of NEM to avoid undesirable modifications on other free sulfydryls during sample preparation, after which non-reduced immunoblot was performed. oATG4B, oligomeric ATG4B. (B) HEK293 cells stably transfected with Flag-ATG4B were incubated with H2O2 (10 mM) for 1 h, after which the cell lysates were treated with DTT (3 mM) for another 30 min or HEK293 cells stably transfected with Flag-ATG4B were pretreated with 10 mM of NAC followed by H2O2 (10 mM) incubation for 1 h, reaction mixture were resolved for non-reduced immunoblot analysis using anti-FLAG antibody. The oligomeric ATG4B (oATG4B) and monomeric ATG4B (mATG4B) were marked on the left. (C-E) Reaction mixture containing His-ATG4B (4 µg) were terminated with 10 mM of NEM and analyzed by CBB after these procedures respectively: treatment with given concentration of H2O2 at 4℃ for 30 min (C); pretreatment with 5 mM of NEM for 10 min to block all cysteine residues, after which H2O2 (10 mM) was added (D); incubation with 20 mM of DTT for 20 min after H2O2 oxidation (E). (F) Recombinant His-ATG4B or His-ATG4BC292S, His-ATG4BC361S and His-ATG4BC292,361S were treated with incremental H2O2 in the presence or absence of DTT (20 mM), then reaction was stopped by addition of 10 mM of NEM. (G) Contrast between His-ATG4B and His-ATG4B2CS with or without NEM alkylation. (H) His-ATG4B and His-ATG4BC292,361S treated with H2O2 (10 mM) with or without DTT (20 mM) incubation were separated in 2D-diagonal SDS-PAGE. BSA was represented as positive control. (I) untreated His-ATG4B; (ii) oxidized His-ATG4B; (iii) oxidized His-ATG4B followed by reduction; (iv) untreated His-ATG4B2CS; (V) oxidized His-ATG4B2CS; (vi) oxidized His-ATG4BC292,361S followed by reduction. The horizontal direction is performed by non-reduced PAGE, with the vertical direction reduced.