Fig. 1.

Paxillin interacts with the N-terminus of kindlin-1 and enhances integrin αIIbβ3 activation. (A) The subdomains of kindlins and their binding partners. (B) PXN, KRT18 and TRIOBP were constructed as Flag-tagged molecules and expressed in CHO-αIIbβ3 cells together with DsRed-fused talin head (TH) and EGFP-fused kindlin-1 (K1) by transient transfection. The transfected cells positive for both DsRed and EGFP were gated for further analysis. (C) The expression levels of Flag-tagged PXN, KRT18 and TRIOBP in the transfected cells were measured by western blotting (WB). (D) Integrin αIIbβ3 activation was expressed by normalized PAC-1 binding, as defined in Materials and Methods. (E) CHO-αIIbβ3 cells were transfected with the siRNA duplexes targeting PXN (siPXN001 and siPXN003) or a control siRNA duplex (siControl). Their efficiencies for knockdown of endogenous PXN were evaluated by western blotting. (F) Control and specific siRNA duplexes targeting for PXN were co-transfected in CHO-αIIbβ3 cells with TH and K1, as indicated, and their effects on integrin αIIbβ3 activation evaluated by the PAC-1 binding assay. Results in D and F represent the mean±s.d. of five experiments; **P<0.01.