Fig. 4: Interaction of hAtg3 F296 residue with the membrane is indispensable for LC3B conjugation and autophagic flux in vivo.
a Atg3 knockout (Atg3−/−) mouse embryonic fibroblasts (MEFs) stably expressing mCherry-EV (empty vector), mCherry-hAtg3, mCherry-hAtg3F296L, or mCherry-hAtg3F296S were cultured in complete media with 100 nM bafilomycin A1 for 3 h and subjected to immunoblotting for mCherry. b Representative immunoblot (n = 4 blots). MEF cells were cultured in complete media (CM) or starvation media (SM) with/without 100 nM bafilomycin A1 (BafA1) to block LC3B-II (LC3B-PE) degradation for 3 h and subjected to immunoblotting with indicated antibodies. c Quantitative analysis of the relative LC3B-II level (n = 4 blots) in in vivo LC3B lipidation experiments. d Quantitative analysis of induced autophagic flux (n = 4 blots). Statistical analysis was performed using one-way ANOVA test followed by Turkey’s multiple comparisons test. In c, data are presented as mean ± SD, P values: ****P < 0.0001, ***P = 0.0001, 0.0002, 0.0001, 0.0001 (left to right, bottom to top); **P = 0.0021, 0.0017, 0.002, 0.0052, and 0.0034 (left to right, bottom to top). In d, data are presented as mean ± SD, P values: *P = 0.0125, 0.0283, and 0.0118 (bottom to top), **P = 0.0053. Source data are provided as a Source Data file.
