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. Author manuscript; available in PMC: 2016 Mar 2.
Published in final edited form as: Curr Biol. 2015 Feb 5;25(5):546–555. doi: 10.1016/j.cub.2014.12.049

Figure 2. Rph1 negatively regulates autophagy.

Figure 2

(A) Autophagy as measured by the Pho8Δ60 assay is increased in rph1Δ and gis1Δ rph1Δ cells. Wild-type (YTS158, BY4742), rph1Δ (YAB300), gis1Δ (YAB301), gis1Δ rph1Δ (YAB302) and atg7Δ (YAB292) cells were grown in YPD (+N) and then starved for nitrogen (−N) for the indicated times. The Pho8Δ60 activity was measured and normalized to the activity of wild-type cells after starvation, which was set to 100% at each time point. The data represent the average of 3 independent experiments; error bars indicate standard deviation.

(B) Autophagy as measured by the GFP-Atg8 processing is increased in rph1Δ and gis1Δ rph1Δ cells. Wild-type (YTS158, BY4742), rph1Δ (YAB300) and gis1Δ rph1Δ (YAB302) cells transformed with a CEN plasmid carrying a GFP-Atg8 construct were grown in rich selective medium and then starved for 1 and 3 h. Cells were collected and protein extracts were analyzed by western blot with anti-YFP antibody and anti-Pgk1 (loading control) antiserum.

(C) Quantification of (B). The ratio free GFP:GFP-Atg8 was measured and normalized to that of wild-type cells after 3 h starvation, which was set to 100%. Average values ± standard deviations of 2 independent experiments are indicated.