Constitutive Rap1 activation and spontaneous VLA-4-mediated cell tethering and arrest on VCAM-1 are impaired in LAD EBV B cells. (A) GTP-bound Rap1 in control and LAD EBV-transformed cells was detected with pull-down assays using GST-RalGDS-RBD (top). Total Rap1 levels were detected with anti-Rap1A (middle). (Bottom) Intensity levels of Rap1 GTP (▪) and total Rap1 (□) were assessed by an image analyzer and are shownin arbitrary units (AU). Rap1 GTP of LAD cells was reduced to 34 ± 7% of that in control cells (mean ± SD of 3 independent determinations). (B) VLA-4-mediated cell capture (transient tethers), rolling, and arrest on VCAM-1 are defective in LAD EBV (L) lymphocytes compared with control EBV cells (c). The categories of different tethers are expressed as percent of the total cells in direct contact with the adhesive substrates. White, gray, and black boxes represent transient, rolling, and arrest categories, respectively. Results are given as mean ± range of determinations in 2 independent experiments. (Inset) Enlarged graph showing categories of cells interacting with the lowest VCAM-1 concentration (0.2 μg/mL). Most of the adhesive interactions were blocked with the α4-specific mAb HP1/2, both on this low-density (shown) as well as on the higher VCAM-1 (not shown) substrates. (C) VLA-4-mediated tethering (□) and arrest (▪) to different densities of surface-bound α4-specific mAb HP1/2 are normal in LAD cells. Shown is 1 representative experiment of 3. Bio1211 at 1 to 10 ng/mL did not interfere with either type of tether to the surface-bound mAb in either control or LAD cells (not shown).