Figure 1. Preparation of cytosolic and... | Rockefeller University Press

Figure 1.

$Figure 1. Preparation of cytosolic and vacuolar fractions. (A) Schematic of the experimental setup: crude vacuolar, autophagosomal, and cytosolic fractions are individually prepared from yeast cells. Incubation of these three fractions together with an energy regeneration system allows the fusion of autophagosomes with vacuoles in vitro. (B) Vacuoles were isolated from Sna3-mCherry Pgk1-BFP atg15Δ cells. Logarithmically growing cells were harvested and lysed, and vacuoles were separated from the cytosolic fraction by a 6,000 g spin. The pellet containing the vacuoles was then further separated on a 0–4–8–15% Ficoll step gradient, and vacuoles were collected at the 0–4% Ficoll interface. The purification steps were analyzed by fluorescence microscopy. DIC, differential interference contrast. Bar, 5 µm. (C) The individual fractions from B were analyzed by anti-Vph1, anti-Pgk1, anti-Pho8, anti-Vti1, and anti-Atg8 Western blotting. One representative experiment out of three is shown. (D) Cytosol was prepared from WT cells treated with 220 nM rapamycin for 4 h by freezer milling and centrifugation at 20,000 g. The supernatant was further spun at 100,000 g. Fractions were analyzed by anti-Vph1, anti-Tom70, anti-Pgk1, anti-Vti1, and anti-Atg8 Western blotting. One representative experiment out of three is shown. Molecular masses are given in kilodaltons.$

Preparation of cytosolic and vacuolar fractions. (A) Schematic of the experimental setup: crude vacuolar, autophagosomal, and cytosolic fractions are individually prepared from yeast cells. Incubation of these three fractions together with an energy regeneration system allows the fusion of autophagosomes with vacuoles in vitro. (B) Vacuoles were isolated from Sna3-mCherry Pgk1-BFP atg15Δ cells. Logarithmically growing cells were harvested and lysed, and vacuoles were separated from the cytosolic fraction by a 6,000 g spin. The pellet containing the vacuoles was then further separated on a 0–4–8–15% Ficoll step gradient, and vacuoles were collected at the 0–4% Ficoll interface. The purification steps were analyzed by fluorescence microscopy. DIC, differential interference contrast. Bar, 5 µm. (C) The individual fractions from B were analyzed by anti-Vph1, anti-Pgk1, anti-Pho8, anti-Vti1, and anti-Atg8 Western blotting. One representative experiment out of three is shown. (D) Cytosol was prepared from WT cells treated with 220 nM rapamycin for 4 h by freezer milling and centrifugation at 20,000 g. The supernatant was further spun at 100,000 g. Fractions were analyzed by anti-Vph1, anti-Tom70, anti-Pgk1, anti-Vti1, and anti-Atg8 Western blotting. One representative experiment out of three is shown. Molecular masses are given in kilodaltons.