Figure 1

Efficient autophagosome targeting of RavZ(ΔCA)-GFP. (A) Representative Western blots of endogenous LC3B in cells expressing 3xFlag-RavZ protein or 3xFlag-RavZ_C258S catalytic mutant in HEK293T cells upon autophagy induction (100 nM rapamycin). Extended blot images including these data are presented in Supplementary Fig. 5A. (B) Schematic diagram of GFP-fused RavZ mutant protein (RavZ(ΔCA)-GFP) (upper) and confocal images depicting the cellular localization of RavZ(ΔCA)-GFP co-expressed with mRFP-LC3B or mRFP-GABARAP in MEF cells treated with 100 nM rapamycin (rapa) + 10 mM NH₄Cl. Scale bar, 10 µm. (C) The bar graphs illustrate the percentages of mRFP-LC3B- or mRFP-GABARAP-positive RavZ(ΔCA)-GFP spots (n = 25 for each group). (D,E) Confocal images showing cellular localization of GFP, GFP-LC3B or RavZ(ΔCA)-GFP with Lysotracker into MEFs upon 100 nM rapa treatment. The bar graph illustrates the ratios of LysoTracker-positive RavZ(ΔCA)-GFP spots number per cell (n = 25 for each group). The data are presented as the mean ± SEM. ***P < 0.001, according to one-way ANOVA followed by Tukey’s post-hoc test. Scale bar, 10 µm. (F,G) Autophagic flux indicates differences in the levels of LC3-II of GABARAP-II in the presence and absence of chloroquine (CQ). The bar graphs illustrate the level of LC3-II or GABARAP-II. The levels of LC3-II and GABARAP-II in the GFP- or RavZ(ΔCA)-GFP-expressing cells were normalized to that of actin in HEK293T cells expressing GFP or RavZ(ΔCA)-GFP. The data are presented as the mean ± SEM of five independent experiments. Extended blot images including these data are presented in Supplementary Fig. 5B. RAP, GABARAP.