American Society of Hematology

Fig. 4.

$Fig. 4. P-selectin triggers CD18 redistribution to Triton X-100–insoluble cytoskeletal fraction and colocalization of Mac-1 clusters with F-actin patches at the site of cell-cell contact. / PMNLs were incubated with platelets (A) or 10 μg/mL P-selectin–IgG chimera (B) for different times in standard conditions. Samples were lysed with CSK buffer, and proteins associated with the Triton X-100–insoluble cytoskeletal fraction were subjected to 6% SDS-PAGE and transferred to Immobilon membrane. The presence of CD18 was analyzed by Western blotting using the anti-CD18 antibody KIM127. The figure shows the results representative of 5 different experiments. (C) PMNLs were incubated for 2 minutes in standard conditions in the absence (i-iii) or in the presence of P-selectin–IgG chimera (iv-vi). After incubation, CD11b and F-actin were stained using the anti-CD11b antibody OKM10, followed by goat anti–mouse Alexa Fluor 488 antibody and rhodamine-phalloidin, respectively, and processed for confocal laser scanning microscopy. Images represent confocal micrographs from the middle third of a single unstimulated cell (i-iii) and of an aggregate of 3 cells (iv-vi). Panels i and iv show F-actin staining, and ii and v show CD11b staining. In the overlay (iii, vi), yellow represents colocalization of the 2 stainings. The figure reports the results of a representative of 5 different experiments.$

P-selectin triggers CD18 redistribution to Triton X-100–insoluble cytoskeletal fraction and colocalization of Mac-1 clusters with F-actin patches at the site of cell-cell contact.

PMNLs were incubated with platelets (A) or 10 μg/mL P-selectin–IgG chimera (B) for different times in standard conditions. Samples were lysed with CSK buffer, and proteins associated with the Triton X-100–insoluble cytoskeletal fraction were subjected to 6% SDS-PAGE and transferred to Immobilon membrane. The presence of CD18 was analyzed by Western blotting using the anti-CD18 antibody KIM127. The figure shows the results representative of 5 different experiments. (C) PMNLs were incubated for 2 minutes in standard conditions in the absence (i-iii) or in the presence of P-selectin–IgG chimera (iv-vi). After incubation, CD11b and F-actin were stained using the anti-CD11b antibody OKM10, followed by goat anti–mouse Alexa Fluor 488 antibody and rhodamine-phalloidin, respectively, and processed for confocal laser scanning microscopy. Images represent confocal micrographs from the middle third of a single unstimulated cell (i-iii) and of an aggregate of 3 cells (iv-vi). Panels i and iv show F-actin staining, and ii and v show CD11b staining. In the overlay (iii, vi), yellow represents colocalization of the 2 stainings. The figure reports the results of a representative of 5 different experiments.

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