Figure 3.

cPLA₂α binding to αIIbβ3 is indirect and depends on αIIb and β3 cytoplasmic tails. Neutravidin beads coated with the indicated integrin cytoplasmic tail model proteins were incubated with platelet lysate or purified His₆-cPLA₂α as described in “Methods.” (A) cPLA₂α was detected by Western blotting. Equal loading of recombinant integrin tails was determined by Coomassie staining. This experiment was performed 3 times. (B) The His₆-cPLA₂α pull-down assay (first 4 lanes) was performed by incubating 20 μg of purified recombinant His₆-cPLA₂α with the indicated integrin tails bound to neutravidin beads. The presence of His₆-cPLA₂α in the pull-down was assayed by immunodetection using an anti-His₆ antibody. Simultaneously, His₆-cPLA₂α was tested for its ability to interact with αIIbβ3 from human platelet lysate in an immunoprecipitation assay (IP) using either an irrelevant isotype match control antibody (IgG) or an anti-His₆ antibody (His₆; last 2 lanes). Equal loading of recombinant tails was determined by Coomassie staining. This experiment was performed twice.