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. 2023 Jul 11;325(3):G265–G278. doi: 10.1152/ajpgi.00053.2023

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Published by the American Physiological Society.

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Figure 4. — Ethanol enhances pancreatic ATG4B activity and complex formation with LC3. To assess ATG4B enzymatic activity, cytosolic fractions obtained from pancreata of untreated mice (A) and mice subjected to indicated treatments were preincubated (or not) with cysteine peptidase inhibitor NEM (10 mM) followed by incubation with LC3 vinyl sulfone (LC3-VS) active site-directed probe (B and C). The levels of ATG4B protein (∼45 kDa; numbers on the right) and its covalent adduct with LC3-VS probe (∼65 kDa) were measured by IB using anti-ATG4B antibody. Lactate dehydrogenase (LDH) served as loading control. C: ATG4B activity was assessed by densitometry of the ATG4B:LC3-VS adduct IB band. D: LC3 was immunoprecipitated from pancreas of mice subjected to indicated treatments; LC3 and ATG4B levels in the immunoprecipitates (IP) were analyzed by IB and quantified by densitometry. Numbers on the bottom show densitometric band intensities normalized to those for control (i.e., no EtOH, no CER) mice. In C, values are means ± SE from three mice per condition. ***P < 0.001; one-way ANOVA with Tukey’s multiple comparison test. CER, cerulein; EtOH, ethanol; IB, immunoblot; LC3, microtubule-associated protein-1 light chain 3; NEM, N-ethylmaleimide.