Figure 1.
Rab2 is degraded via autophagy pathway. (A and B) U2OS cells were starved in EBSS medium for (0, 1, 2, 3, 4, or 5 h), and the levels of endogenous Rab2 and p62 were measured by western blot (A) and quantified in B. Data are shown as means ± SEM and analyzed with one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. (C and D) Rab2 wild type (WT) cells, Rab2 knockout (KO) U2OS cells, and Rab2 KO U2OS cells that transiently expressing 1 µg GFP-Rab2, cells were treated with EBSS or EBSS and Bafilomycin A1 for 2 h. Quantification of cleavage GFP is shown in D. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (E and F) HEK293T cells transiently expressing GFP-Rab2 or GFP-LC3 were treated with Torin1 for 0, 2, or 4 h. Quantification of cleavage GFP is shown in F. Data are shown as means ± SEM and analyzed with one-way ANOVA. *P < 0.05, ***P < 0.001. (G and H) HEK293T cells transiently expressing GFP-Rab2 WT, GFP-Rab2 Q65L, or GFP-Rab2 N119I were treated with Torin1 for 0, 2, or 4 h. Quantification of cleavage GFP is shown in H. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (I and J) Atg7 WT or Atg7 KO HEK293 cells transiently expressing GFP-Rab2 were treated by Torin1 for 0, 2, or 4 h. Quantification of cleavage GFP is shown in J. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (K and L) HEK293T cells transiently expressing GFP-Rab2 WT, GFP-Rab2 mutant (Cys211, 212Ser) were treated with Torin1 for 0, 2, or 4 h. Quantification of cleavage GFP is shown in L (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (M and N) Atg7 WT or Atg7 KO U2OS cells transiently expressing mCherry-GFP-Rab2 were treated with Torin1 for 2 h and were analyzed by confocal microscopy for mCherry+GFP− puncta. mCherry+GFP− puncta were quantified in N (n = 30 cells per group). Scale bars, 10 μm. The scale bars in the magnification boxes are 2 µm. Data are shown as means ± SEM, and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (O and P) U2OS cells transiently expressing mCherry-GFP-Rab2 or mCherry-GFP-LC3B, cells were treated with Torin1 for 2 h, staining with LysoTracker Blue DND-22, analyzed by confocal microscopy for mCherry+GFP−LysoTracker+ puncta and quantified in P (n = 30 cells per group). Scale bars, 10 μm. The scale bars in the magnification boxes are 2 µm. Data are shown as means ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (Q and R) Atg7 WT or Atg7 KO HEK293 cells were treated with Torin1 for 0, 2, 4, 6 h and the levels of endogenous Rab2, p62, LC3 and Atg7 were detected by western blot and quantified in R. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F1.

Rab2 is degraded via autophagy pathway. (A and B) U2OS cells were starved in EBSS medium for (0, 1, 2, 3, 4, or 5 h), and the levels of endogenous Rab2 and p62 were measured by western blot (A) and quantified in B. Data are shown as means ± SEM and analyzed with one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. (C and D) Rab2 wild type (WT) cells, Rab2 knockout (KO) U2OS cells, and Rab2 KO U2OS cells that transiently expressing 1 µg GFP-Rab2, cells were treated with EBSS or EBSS and Bafilomycin A1 for 2 h. Quantification of cleavage GFP is shown in D. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (E and F) HEK293T cells transiently expressing GFP-Rab2 or GFP-LC3 were treated with Torin1 for 0, 2, or 4 h. Quantification of cleavage GFP is shown in F. Data are shown as means ± SEM and analyzed with one-way ANOVA. *P < 0.05, ***P < 0.001. (G and H) HEK293T cells transiently expressing GFP-Rab2 WT, GFP-Rab2 Q65L, or GFP-Rab2 N119I were treated with Torin1 for 0, 2, or 4 h. Quantification of cleavage GFP is shown in H. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (I and J) Atg7 WT or Atg7 KO HEK293 cells transiently expressing GFP-Rab2 were treated by Torin1 for 0, 2, or 4 h. Quantification of cleavage GFP is shown in J. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (K and L) HEK293T cells transiently expressing GFP-Rab2 WT, GFP-Rab2 mutant (Cys211, 212Ser) were treated with Torin1 for 0, 2, or 4 h. Quantification of cleavage GFP is shown in L (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (M and N) Atg7 WT or Atg7 KO U2OS cells transiently expressing mCherry-GFP-Rab2 were treated with Torin1 for 2 h and were analyzed by confocal microscopy for mCherry+GFP− puncta. mCherry+GFP− puncta were quantified in N (n = 30 cells per group). Scale bars, 10 μm. The scale bars in the magnification boxes are 2 µm. Data are shown as means ± SEM, and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (O and P) U2OS cells transiently expressing mCherry-GFP-Rab2 or mCherry-GFP-LC3B, cells were treated with Torin1 for 2 h, staining with LysoTracker Blue DND-22, analyzed by confocal microscopy for mCherry+GFP−LysoTracker+ puncta and quantified in P (n = 30 cells per group). Scale bars, 10 μm. The scale bars in the magnification boxes are 2 µm. Data are shown as means ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (Q and R) Atg7 WT or Atg7 KO HEK293 cells were treated with Torin1 for 0, 2, 4, 6 h and the levels of endogenous Rab2, p62, LC3 and Atg7 were detected by western blot and quantified in R. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F1.

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