non radioactive ceramide standard  (Avanti Polar)

Journal: eLife

Article Title: Doxorubicin blocks proliferation of cancer cells through proteolytic activation of CREB3L1

doi: 10.7554/elife.00090

Figure Lengend Snippet: Figure 7. Doxorubicin-induced synthesis of ceramide stimulates cleavage of CREB3L1. (A),(C) On day 0, Huh7 cells were seeded at 4 × 105 per 60-mm dish. On day 1, the cells were treated with indicated concentrations of myriocin (A) or fumonisin B1 (C) for 2 hr, followed by co-incubation with 200 nM doxorubicin. On day 2, 24 hr after the doxorubicin treatment, cells were analyzed for cleavage of CREB3L1 by immunoblot analysis as described in Figure 1B. (B) Huh7 cells treated with or without 30 µM myriocin for 2 hr followed by co-treatment with doxorubicin were analyzed as described in Figure 3A. (D) Huh7 cells treated with 10 µM C6-ceramide for 3 hr were analyzed as described in Figure 6B. Results are reported as mean ± S.E.M. of triplicate incubations from a representative experiment. (E) Huh7 cells treated with indicated concentration of C6-ceramide for 24 hr were analyzed as described in Figure 1B. (F) Indicated cells treated with indicated concentration of C6-ceramide for 48 hr were analyzed as described in Figure 3A. (B),(F) Results are reported as mean ± S.E.M. of three independent experiments. DOI: 10.7554/eLife.00090.009

Article Snippet: Lipid extracts were mixed with 50 μg of non-radioactive ceramide standard (Avanti Polar Lipids) and analyzed by Thin Layer Chromatography (TLC) on POLYGRAM SIL G plates in a solvent system of chloroform/acetate (90:10; vol/vol) for ceramide separation.

Techniques: Incubation, Western Blot, Concentration Assay