Figure 3. Low UBB+1 expression activates autophagy. (A–B) Western blot of GFP-Atg8p processing into free GFP. GAPDH was used as the loading control. (C–D) Translocation of GFP-Atg8p into yeast vacuole. Top panel: images from FLUO-GFP filter. Bottom panel: images from DIC filter. White arrow: GFP fluorescence inside vacuole. Scale bar = 5 μm. (E) The ratio of free GFP to total GFP (uncleaved GFP-ATG8 + free GFP) under wild type background was calculated and presented based on (A). Data is shown as average values ± SD from biological triplicates. (F) The percentage of cells with diffuse vacuolar GFP fluorescence was counted and represented based on (C–D). Above 200 cells were count per sample (n = 3 ± SD). The asterisk (*) indicates a statistically significant p-value of < 0.05 from untreated control strain.