Protein Extraction and Immunoblot Analysis

Cells were harvested and lysed in 1 ml 0.2 M NaOH and 0.2% β-mercaptoethanol. Proteins were precipitated via the addition of 100 µl TCA and centrifuged at 12,000 rpm. Proteins were resuspended in 120 µl 2× SDS sample buffer followed by the addition of 30 µl of 1 M Tris base, pH ∼11. Samples were heated at 75°C and analyzed via immunoblot (Peng and Weisman, 2008 (link)). For immunoblot analyses, mouse anti-GFP (1:1,000; Roche), rabbit anti-TAP (1:1,000; Thermo Fisher Scientific), mouse anti-Pgk1 (1:10,000; Invitrogen), sheep anti-Vac17 (1:1,000; custom made; 21st Century Biochemicals), rabbit anti–phospho-Thr240 (1:2,500; custom made; 21st Century Biochemicals), and rabbit anti–phospho-Ser222 (1:2,500; custom made; 21st Century Biochemicals) antibodies were used (see the Antibody preparation section).