Figure 1. Verification of the cleavage of FRET substrates by Atg4 using SDS-PAGE. (A) Schematic representation of the FRET-LC3B and FRET-GATE-16 constructs. The cleavage site of LC3B and GATE-16 at the glycine (G) residue is indicated. (B and C) FRET-LC3B, FRET-LC3B^G120A (B) or FRET-GATE-16 (C) (5 µg) were incubated with Atg4B, Atg4B^C74S or Atg4A (0.25 µg), respectively. The reaction was stopped at different times using SDS-PAGE sample buffer. The substrates and the cleaved products were separated by SDS-PAGE and examined by CBB staining. Representative gel images from three independent experiments are shown in the left panels. Percentage of cleaved substrate was calculated based on the densitometry of protein bands of the representative experiments (right panels).