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. 2017 Feb 16;8(12):20067–20085. doi: 10.18632/oncotarget.15379

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Copyright: © 2017 Stephan et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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a. Merged confocal microscopic images of SKW6.4 cells stimulated with 100 ng/ml SFF-CD95L. The CD95L was visualized by an anti-FLAG antibody and anti-mouse Alexa-Fluor 488. A-SMase stained by a polyclonal anti-A-SMase antibody and anti-rabbit Alexa-Fluor 555 was detected in CD95-receptosomes between 1 min and 60 min internalization. Co-localization of CD95 and A-SMase is indicated in yellow. b. Magnetic and non-magnetic fractions were used to measure A-SMase activation in SKW6.4 cells after indicated time points of CD95 internalization. Three independent preparations were pooled and measured in triplicate. (1way-Anova with Bonferroni's multiple comparison test) c. Western blot analysis of CD95 magnetic and non-magnetic fractions with anti-CD95 and anti-A-SMase antibodies. Corresponding to the activity assay, Western blot analysis demonstrated a biphasic appearance of A-SMase in the magnetic fractions.