Research Paper Volume 15, Issue 19 pp 9965—9983

Figure 4. The changes in the level of autophagy markers during CLS assay. (A–D) The level of autophagy markers in the strains carrying fluorescently tagged GFP-Atg8 (YTE17) (A), Nvj1-GFP (YTE18) (B), Vac8-GFP (YTE19) (C), and Atg39-GFP (YTE20) (D) measured by flow cytometry. The mean from three independent biological repetitions is shown. (E) The level of GFP-Atg8 and its degradation product, free GFP, in the YTE17 strain, visualized by immunoblotting with anti-GFP antibody. One of three independent biological replicates is shown. (F, G) The quantification of western blot results. (F) The level of GFP-Atg8 and its degradation product GFP was normalized to the Pgk1 level and expressed relative to the GFP-Atg8 level at time 0 (protein level in the exponential growth phase). (G) The graph shows the GFP/GFP-Atg8 ratio at various time points. Data for each time point was normalized to the Pgk1 level. The mean of three biological replicates is shown for each time point. Bars indicate standard deviations. Statistical significance with respect to time 0 was assessed using the Student T-test (* p < 0.05; ** p < 0.01; *** p < 0.001: NS - non-significant).