Interaction of vWF with soluble S. aureuscomponents or with SPA.

(A) Western ligand and Western blot analyses of whole-cell staphylococcal lysates. S. aureus Cowan 1, Newman, and NCTC 8325-4 lysates (2 μL) (lanes 1, 2, and 3, respectively) were separated on 7.5% SDS-PAGE, blotted on nitrocellulose, and incubated either with [DIG]-vWF in PBS (left), with polyclonal [DIG]-anti-SPA-Abs in PBS (right), or with PBS alone (center). Binding was detected using anti-DIG-Fab fragments and subsequent exposure with a chromogenic substrate (5 minutes). (B) Western ligand analysis of vWF binding to lysates of S. aureus wild-type (lanes 1, 2, and 3, respectively) and their respective Δspa deletion mutants (lanes 1′-3′). Experimental conditions identical to those in A (left). For enhanced detection sensitivity, gels were intentionally overloaded (20 μL lysate/lane), and blots were overexposed (30 minutes). (C) SDS-PAGE of rhvWF and Western ligand analysis of SPA binding. rhvWF expressed from Chinese hamster ovary cells (40 μg) was subjected to SDS-PAGE (7.5% gels), and gels were either stained with Coomassie blue (left) or used for blotting of vWF onto nitrocellulose (right). Membranes were subsequently blocked and incubated with [DIG]-rSPA, and rSPA binding was demonstrated in a color reaction described in A.