Fig 1: ATG4A is cleaved by viral protease 2A before glycine 374. (A) In vitro cleavage assay was performed with no protease control, purified viral protease 3C (left) or 2A (middle) in the presence of Flag-ATG4A. SNAP29 and eIF4G were used as a positive control for 3C and 2A activity, respectively. Membranes from 2A in vitro cleavage (middle) were stained with Ponceau S Stain or immunostained with anti-HSP90 antibody to probe total protein and alternative loading control respectively. (B) Illustration of the potential cleavage sites in the open reading frame of ATG4A. Estimated size of Flag-tagged fragments was calculated by adding 3×Flag (2.7 kDa) to size of potential N-terminal cleaved fragments (estimated via Expasy Compute pl/Mw tool). Predicted fragment size consistent with cleavage is highlighted (C) HEK293T cells were transfected with GFP, ATG4WT, or ATG4AG374E for 24 h, followed by sham or CVB3 infection (MOI = 100) for 8 h. Cell lysates were harvested and probed for FLAG-ATG4A using anti-Flag antibody, VP1 and ACTB. (D) Schematic diagram of the function domains of Flag-ATG4A, identified cleavage site by 2A, and the sizes of cleavage fragments. Sequence alignments between ATG4A and ATG4B within the cleavage region of the fragments are shown. LIR: LC3-interacting region is shown in underlined red text. Results in this figure are representative of two to three independent experiments.