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. 2019 Jun 2;11(6):313. doi: 10.3390/toxins11060313

Figure 1.

Figure 1

Screening for ATG4B inhibitors with medicinal plants using ATG4B cleavage assays. (A) Schematic diagram for the biochemical reaction to measure ATG4B proteolytic activity. Briefly, the substrate GABARAPL2 fused with the N-terminal C-myc tag and C-terminal S-tag. If extracts inhibited ATG4B, both full-length C-myc-tagged GABARAP2 (upper band) and a short peptide of S-tag can be detected by immunoblotting. (B) Recombinant ATG4B (5 nM) was mixed with the GABARAPL2 fusion protein (1 μM) in a reaction buffer containing 50 mM Tris-base, pH 8.0, 150 mM NaCl, and 1 mM DTT with 10-fold diluted formosan plant extracts (10 or 100 μg/mL). The retained S-tag and full-length C-myc tag (upper band) were determined by immunoblotting. The reactions without (+) and with (−) recombinant ATG4B were used as positive and negative controls of inhibition, respectively. (C) The protein levels of S-tag and (D) full-length C-myc-tagged GABARAPL2 were quantitated by ImageJ and normalized by positive control (+) as 100% inhibition of ATG4B in each blot.