FIGURE 6.

Effects of depletion of SM versus ceramide generation on ERM proteins. A, HeLa cells were cultured in FBS-free medium for 2 h and treated with bSMase (25 milliunits/ml) or SMase D (300 milliunits/ml) to cause the same fold decrease in SM species for 10 min, and ceramide (Cer) and ceramide 1-phosphate were monitored. B, HeLa cells were treated with bSMase or SMase D as before and phospho-ERM, and total ezrin was measured by Western blotting. C, HeLa cells were treated with bSMase (10 min), SMase D (10 min), or with SMase D (10 min), and then with bSMase (10 min) and stained against phospho-ERM (green), actin filaments (stained with rhodamine phalloidin, red) and nuclei (stained with Draq5, blue). D, HeLa cells were treated with bSMase or SMase D as in A, and the presence of SM was monitored by lysenin-Venus (green channel). Cells were fixed, and the nonpermeabilized (upper panels) or permeabilized (lower panels) cells were analyzed by confocal microscope. Nuclei were visualized with Draq5 (blue) and in permeabilized cells F-actin was visualized with rhodamine phalloidin staining (red). tEzrin, total ezrin.