Detection of SNF7 in MVEs and isolated vacuoles. (A and B) Immunolabeling of SNF7 on MVEs from high-pressure–frozen/freeze-substituted root cells. (C) The area indicated by a yellow box in B shown at higher magnification. Red arrowheads indicate gold particles. (D) Immunoblot detection of SNF7, cBPPase (cytoplasmic control), and H⁺PPase (vacuolar membrane control) in protein extracts from isolated protoplasts and vacuoles of wild-type, lip5, and atg7 mutant seedlings. Images of isolated protoplasts and vacuoles are shown at the top. Bars: (A–C) 50 nm; (D) 5 µm. (E) Protease protection assay. Isolated vacuoles were incubated in proteinase K (PK) for 1 h with or without 1% Triton X-100 (TX). SNF7 and H⁺PPase were detected by immunoblotting. Molecular masses are indicated in kilodaltons.