Cer, C1P, and Ceramide kinase activity determination

Cer and C1P measurements were achieved as described in Bini et al. [39]. Briefly, cells were labeled with 40 μCi/ml of [³H] palmitate (Perkin Elmer, Connecticut, USA) for 24h in serum–free medium in the presence or absence of agonists. Cells were then washed with ice-cold calcium-free phosphate-buffered saline (PBS) and scraped into methanol and chloroform (2:1) (Sigma Aldrich, Milan, Italy). Lipids were extracted by separation of phases as described in [39]. Chloroform phases were dried, and lipids were separated by thin-layer chromatography (TLC) using silica gel 60-coated glass plates (Merck, Darmstadt, Germany). The plates were developed for 50% of their lengths with chloroform/methanol/acetic acid (9:1:1, v/v/v) and then dried. They were then developed for their full length with petroleum ether/diethylether/acetic acid (60:40:1, v/v/v) (Sigma Aldrich, Milan, Italy). The position of Cers was identified after staining with I₂ vapor by comparison with authentic standards. Radioactivity of the samples, obtained by scraping the Cer and C1P spots from the plates, was quantified by liquid scintillation counting. CerK activity was assayed as described previously [39, 47] with some modifications.