Fig. 3

ESCRT is required for microautophagy after nitrogen starvation. a Cells of strains SCU2684 (wild-type; BY4741), SCU5456 (vps27∆), SCU6187 (vps28∆), SCU6188 (vps36∆), and SCU4337 (vps24∆) harboring plasmid pSCU2366 (pGFP-PHO8) pretreated with FM4–64 were transferred to nitrogen-depleted medium (SD-N) for 6 h. Fluorescence micrographs are shown. Scale bars, 5 μm. b The same strains as used in panel a was similarly treated with nitrogen starvation. Whole cell extracts were subjected to western blotting using an anti-GFP antibody. Pgk1 was detected as a loading control using an anti-Pgk1 antibody. Free GFP processed from GFP-Pho8 nitrogen starvation was measured, and quantified by calculating the ratio of cleaved free GFP versus the sum of uncleaved GFP-Pho8 and free GFP. The average (± standard deviation) was determined from three independent experiments, and relative values were normalized against the value in control cells are shown. For group comparisons, p-values were calculated using two-way ANOVA with Bonferroni correction. **, p < 0.001