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Fig. 2. Role of the RavZ LIR motifs on the delipidation of LC3B or GARARAP protein in rapamycin-induced autophagy. (A, B) Confocal images (A) showing cellular localization of GFP-LC3B or GFP-GABARAP, co-expressed with 3xFLAG-RavZ protein or the indicated RavZ mutants in MEFs upon autophagy induction (100 nM rapamycin (Rapa) + 50 μM CQ, 4 h). Arrows are representative of autophagic membranes. Scale bar: 10 μm. The bar graphs (B) illustrate the autophagosome spot numbers per cell (n = 20 for each group). 3xFLAG-RavZ and 3xFLAG-RavZmLIR1/2–3 reduce the number of GFP-LC3B or GFP-GABARAP-positive autophagic membranes in rapamycin or rapamycin + CQ-treated cells compared to either mRFP or the 3xFLAG-RavZC258S catalytic mutant. Data are presented as mean ± SEM. N.S., not significant. ***P < 0.001 (one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test). (C) Depletion of endogenous LC3B-II by RavZ protein and the indicated mutants using Western blot analysis in HEK293T cells. Representative Western blots of three independent experiments of endogenous LC3B in cells expressing 3xFLAG-RavZ protein or the indicated mutants in MEFs upon autophagy induction (100 nM rapamycin). 3xFLAG-RavZ or 3xFLAG-RavZmLIR1/2–3 reduces the LC3B-II levels more than does either GFP or 3xFLAG-RavZC258S.
BMB Reports 2019;52:700~705
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