3h serine

To measure the conversion of ceramide to IPC, cells were labeled with [³H]serine (American Radiolabeled Chemicals, Inc.) as previously described (Kajiwara et al., 2008 (link), 2014 (link)) with the following modifications. In brief, 20 µCi [³H]serine was added to 10 OD₆₀₀ of cultures in mid-logarithmic growth phase in SC without serine at 25°C, and the cells were grown for 1 h. For experiments with strains containing the temperature-sensitive sec18-1 allele, cells were initially grown at 25°C, 200 µg/ml cycloheximide was added to the medium, the cells were grown for 20 more minutes at 25°C, the culture was shifted to 37°C for 30 min, and then labeled for 30 min with 20 µCi [³H]serine (Funato and Riezman, 2001 (link)). After labeling, growth was stopped by addition of 10 mM NaF and 10 mM NaN₃ and placing cultures on ice for 20 min. The cells were washed with water, and lipids were extracted and subjected to mild alkaline hydrolysis to deacylate glycophospholipids as described (Kajiwara et al., 2008 (link), 2014 (link)). Lipids were dried under nitrogen and separated on silica TLC plates (EMD Millipore) using the chloroform/methanol/4.2 N ammonium hydroxide (9:7:2). TLC plates were scanned on a RITA* Thin Layer Analyzer (Raytest) to quantify radiolabeled lipids and determine the ratio of IPC to ceramide.