Yeast strains containing the pRS316-GFP-Atg8 plasmid were cultured in starvation medium at 30 or 37° to induce autophagy. Cells were collected and subjected to trichloroacetic acid precipitation, used to extract proteins from cells as described previously (Yang and Rosenwald 2016). Total proteins from 0.8 OD600 were separated on precast polyacrylamide gels (Any kD, Bio-Rad, 456–9036). The proteins were transferred onto nitrocellulose membranes and the membrane was incubated with a mouse anti-GFP antibody, followed by an HRP-conjugated anti-mouse secondary antibody. HRP signals were visualized using an ECL prime kit (GE Healthcare) and detected with an ImageQuant LAS 4010 imager (GE Healthcare). Each set of western blot experiments was repeated three times. Representative examples are shown in each figure.