Figure 3

ATG7 isoform 2 fails to lipidate LC3/GABARAP. (A) Wild type MEFs or Atg7^−/− MEFs stably expressing FLAG-tagged empty vector (EV), ATG7(1) or ATG7(2) were treated with Bafilomycin-A1 (Baf-A1) or vehicle control (DMSO) for 4 h and stained with FLAG (red) and LC3B (green) antibodies. Representative images of three experiments are shown. Scale bar represents 20 µm and applies to all images. (B) Quantification of LC3B puncta was performed using CellProfiler software. Error bars represent SEM of three independent experiments. Two-way Anova with Sidak’s multiple comparisons test was performed revealing no significant statistical difference. (C) Long-lived protein degradation (LLPD) assay of wild type MEFs or Atg7^−/− MEFs stably expressing FLAG-tagged empty vector (EV), ATG7(1) or ATG7(2), treated with Baf-A1 or DMSO for 4 h. Error bars represent SEM of three independent experiments. Two-way Anova with Sidak’s multiple comparisons test was performed revealing no significant statistical difference. (D) Western blot analysis of lysates from wild type MEFs or Atg7^−/− MEFs stably expressing FLAG-tagged empty vector (EV), ATG7(1) or ATG7(2), treated with Baf-A1 or DMSO for 4 h. Membranes were probed with antibodies against FLAG, p62, LC3A, LC3B and Actin. (E) Quantification of band intensities was performed using ImageJ software. Error bars represent SEM of three independent experiments. Two-way Anova with Sidak’s multiple comparisons test was performed to determine statistical significance of ATG7(1) or ATG7(2) expressing Atg7^−/− MEFs compared to EV Atg7^−/− MEFs. *p < 0.05.