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Fig. 1 | Biology Direct

Fig. 1

From: Rab32 uses its effector reticulon 3L to trigger autophagic degradation of mitochondria-associated membrane (MAM) proteins

Fig. 1

Active Rab32 Promotes Autophagy. A Representative immunofluorescence images of MCF7 cells stably expressing LC3-GFP and transfected with FLAG-tagged wild type (WT), dominant active (Q85L) or dominant negative (T39N) Rab32, probed for FLAG-tagged Rab32 (red) and nuclei (DAPI, blue) in parallel. Bar indicates 15 µm. Quantification of cells with > 10 LC3 puncta, indicating increased autophagy, were expressed as a percent of total cells counted. ≥ 100 cells per condition were counted in each (n = 3) independent experiment **p ≤ 0.001. Inset shows magnification of a representative area, evidencing overlap between Rab32 and LC3-GFP. B Immunoblot of MCF7 cells transfected with pcDNA3 vector as a control, or with Rab32 plasmids as indicated. Blots were probed for LC3, FLAG and Tubulin as a loading control. Densitometric analysis of LC3II/Tubulin on the bottom. n = 3 **p ≤ 0.001. C Representative immunofluorescence images of MCF7 cells stably expressing LC3-GFP with control siRNA or siRNA against Rab32, probed for endogenous Rab32 (red) and nuclei (DAPI, blue) in parallel. Bar indicates 10 µm. Quantification of cells with > 10 LC3 puncta, indicating increased autophagy, were expressed as percent of total cells counted. ≥ 100 cells per condition were counted in each (n = 3) independent experiment. p > 0.05. D Immunoblot of MCF7 cells with control siRNA or siRab32 probed for endogenous Rab32 and LC3. Densitometric analysis of LC3II/Tubulin on the bottom. n = 3, p > 0.05

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