The putative Kindlin-3 binding site on the β tail of VLA-4 is not required for VLA-4 affinity or adhesiveness to VCAM-1. (A) Sequence of β₁ integrin subunit, showing its putative association sites with Kindlin-3, talin1, filamin, and ICAP-1 in leukocytes. The 2 NPXY binding sites are highlighted. (B) Kindlin-3 and talin1 expression in Jurkat A1 cells (β₁ integrin deficient) reconstituted with either wt β₁ (wt), β₁ΔNPIY (ΔNPIY), or β₁ΔNPKY (ΔNPIY). Lysates of each Jurkat transfectant were immunoblotted with anti–Kindlin-3 antibody and anti-talin mAb. (C) FACS staining of β₁ on the various Jurkat A1 transfectants. α₄ expression was identical on all transfectants (data not shown). (D) Frequency and strength of tethers mediated by Jurkat β₁ wt transfectants, Jurkat β₁ΔNPIY, or Jurkat β₁ΔNPKY transfectants perfused over medium density soluble VCAM-1 (370 sites/μm²). All interactions were determined at a shear stress of 0.75 dyn/cm² in 2 fields of view, and results shown are the mean ± range. *P < .01 for firm tethers of the compared experimental groups. (E) Binding of soluble VCAM-1-Fc (at a saturating concentration of 60 μM) to Jurkat β₁ wt cells, Jurkat β₁ΔNPIY, and β₁ΔNPKY transfectants, detected by fluorescence staining. No VCAM-1 binding could be detected in the presence of the VLA-4 blocker, Bio1211 (data not shown). Data are representative of 2 independent experiments.