Ceramide Accumulation Uncovers a Cycling Pathway for the cis-Golgi Network Marker, Infectious Bronchitis Virus M Protein

Maceyka, Michael; Machamer, Carolyn E.

Lipid Synthesis Assays. Cells were seeded onto 6-cm plastic dishes 2 d before the experiment so that they were 90% confluent the day of the experiment. Cells were incubated in DME supplemented with 50 g/ml cycloheximide, and half of the dishes were also incubated with 5 mM ␤ CA. 1 h later, fresh medium containing cycloheximide and [ 3 H]palmitate (100 Ci/ dish) was added, along with either 1% isopropanol (control and ␤ CA) or 100 M PDMP and 5 mM ␤ CA if indicated. 1 h later, cells were trypsinized and washed off the plate in 0.8 ml cold PBS. To normalize lipid levels, 50 l were removed for a protein assay by the method of Bradford (1976) . Lipids were then extracted by the method of Bligh and Dyer (1959) . Labeled samples were doped with 10 g cold ceramide to allow for visualization. Samples were run on 10 ϫ 10 cm high performance thinlayer chromatography plates with a mobile phase of chloroform/glacial acetic acid (9:1; Abe et al., 1992) . Plates were sprayed with water to visualize the ceramide bands that were scraped and counted after the addition of scintillation fluid. Alternatively, plates were dipped in 10% 4,5-diphenyloxazole in chloroform for visualization by fluorography (Henderson and Tocher, 1992) .