Journal: Nature Communications

Article Title: Multifaceted membrane interactions of human Atg3 promote LC3-phosphatidylethanolamine conjugation during autophagy

doi: 10.1038/s41467-023-41243-4

Figure Lengend Snippet: a Left panel: sequence comparison of hAtg3 C-terminal residues 262–277 with the analogous region in selected organisms. Right panel: helical wheel plot for hAtg3 residues 265–277. b Representative SDS-PAGE gel images of time-dependent formation of LC3B-PE for hAtg3 wildtype (WT) and mutants ( n = 3). CL is the control incubated for 180 min without liposomes. Asterisk indicates a small amount of degradation of LC3B in the presence of ATP. c Left panel: sequence comparison of hAtg3 C-terminal residues 291–300 with the analogous region in selected organisms. Right panel: helical wheel plot for hAtg3 residues 291–300. d Representative SDS-PAGE gel images of time-dependent formation of LC3B-PE for hAtg3 mutants ( n = 3). CL is the control incubated for 180 min without liposomes. Asterisk indicates a small amount of degradation of LC3B in the presence of ATP. For b and d , source data are provided as a Source Data file.

Article Snippet: Cells were incubated in complete or starvation media in the absence and presence of 100 nM bafilomycin A1 (BafA1) (ThermoFisher scientific, J61835.MCR) for 3 h and subjected to immunoblotting using the mCherry antibody (Abcam, ab125096) with a ratio of 1:1000, the β-actin antibody (Sigma, A5441-100UL) with a ratio of 1:10,000, and the LC3 antibody (Novus Biologicals, NB100-2220) with a ratio of 1:5000.

Techniques: Sequencing, Comparison, SDS Page, Control, Incubation, Liposomes

Journal: Nature Communications

Article Title: Multifaceted membrane interactions of human Atg3 promote LC3-phosphatidylethanolamine conjugation during autophagy

doi: 10.1038/s41467-023-41243-4

Figure Lengend Snippet: a Atg3 knockout (Atg3 −/− ) mouse embryonic fibroblasts (MEFs) stably expressing mCherry-EV (empty vector), mCherry-hAtg3, mCherry-hAtg3 F296L , or mCherry-hAtg3 F296S 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.

Article Snippet: Cells were incubated in complete or starvation media in the absence and presence of 100 nM bafilomycin A1 (BafA1) (ThermoFisher scientific, J61835.MCR) for 3 h and subjected to immunoblotting using the mCherry antibody (Abcam, ab125096) with a ratio of 1:1000, the β-actin antibody (Sigma, A5441-100UL) with a ratio of 1:10,000, and the LC3 antibody (Novus Biologicals, NB100-2220) with a ratio of 1:5000.

Techniques: Knock-Out, Stable Transfection, Expressing, Plasmid Preparation, Cell Culture, Western Blot, Blocking Assay, In Vivo