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Fig. 5. Moesin association with actin cytoskeleton. The association of moesin with actin cytoskeleton was determined by its solubility in Triton X-100, which preserves the cytoskeleton and the cytoskeleton-associated proteins. Confluent cells were either left untreated (CTR) or stimulated with forskolin (FK) or preincubated for 3 hours with the control peptide or with the ERM peptide or with Y27632, an inhibitor of Rho kinase. Cells were incubated in Triton X-100 buffer and extracted proteins were immunoblotted with moesin antibody (A) or with P-Thr-moesin antibodies (B); on the right of each panel, the densitometric analysis of the bands obtained was shown. Histograms show the ratios between the densitometric signals of P-Thr-moesin and total moesin in each Triton-soluble fraction (C). (*P<0.01, **P<0.05, #P<0.001). Actin depolymerization is accompanied by a dissociation of moesin from actin cytoskeleton with a consistent reduction in P-Thr-moesin abundance. (D) Immunolocalization of P-Thr-moesin in CD8 cells. Cells were either left untreated or stimulated with forskolin. Fluorescence was visualized by epifluorescence microscopy. In untreated cells, P-Thr-moesin was localized at the cell-cell adhesion sites and along intracellular filaments depicting actin stress fibers (arrowheads). This particular staining disappeared in forskolin-stimulated cells. Bars, 5 µm.
