Fig. 4

Crkl and VASP dynamically interact in human platelets. (a) Lysates of human platelets were immunoprecipitated (IP) with Abs against Crkl or isotype control antibodys (control) as described in Methods. Lysate, wash fraction (Wash), and precipitated material were analyzed by Western blotting with anti-VASP (upper panel) or anti-Crkl Abs (lower panel), respectively. The arrows on the right hand side indicate the position of VASP and Crkl. One representative experiment (out of three) is shown. B-E, Washed human platelets were seeded onto fibronectin-coated glass slides for 2 min (b), 5 min (c), 12 min (d), or 30 min (e) before fixation and staining with anti-VASP-specific Abs (green), anti-Crkl-specific Abs (red) or with fluorescent conjugated phalloidin to visualize actin fibers (blue). The top panels in each section show individual stainings; lower panels show co-localization staining of VASP and Crkl, VASP and actin, and Crkl and actin. Black arrows, white arrowheads, and white arrows indicate filopodia-, focal adhesion-, and lamellipodia-like membrane protrusions, respectively. All images have a dimension of 20 × 20 μm to better visualize platelet spreading, scale bar in B is 5 μm. In e´, magnified views of the indicated areas in e are shown; scale bar 1 μm