Inducible translocation of FasL into lipid membrane rafts. (A) HEK293 cells expressing hFasL were cocultured with L1210 cells (no Fas receptor; left panel) or with L1210 cells stably transfected with Fas (right panel) for 80 minutes at 37°C before solubilization in Brij 98 and were subjected to sucrose gradient separation. Single fractions were analyzed by Western blot with anti-FasL, anti-Fyn, and anti-Rab5 antibodies. (B) Samples were taken at different time points from the same coculture experiments described in panel A to quantify cell death in the target cell population (L1210 and L1210 Fas cells) by flow cytometry analysis (sub-G1 content; left panel). In addition, after 80 minutes of coculture, total protein lysates were prepared from an aliquot of the cells. Western blot analysis with the antiphosphotyrosine antibody 4G10 revealed increased protein phosphorylation in the sample derived from Fas receptor–expressing cells, indicating efficient Fas/FasL interaction (right panel).
