Role of PLC and PKC in Rap1 activation and stimulated T-cell adhesion. (A) Western blots of Rap1-GTP in T cells pretreated with a PLC inhibitor (10 μM U73122), a calcium chelator (1 μM BAPTA-AM), or a PKC inhibitor (10 μM Gö 6850) for 10 minutes, and then treated with 100 nM SDF-1α for 10 seconds (left) or 1μg/mL PMA for 5 minutes (right). Active Rap1 was detected by pull-down assay. Vertical lines have been inserted to indicate a repositioned gel lane. (B) Inhibition of PLC or PKC impairs SDF1-stimulated adhesion to ICAM-1 or VCAM-1 under shear flow. T cells were pretreated with 10 μM U73211 or 10 μM Gö 6850 for 10 minutes and perfused over ICAM-1 or VCAM-1 coimmobilized with SDF-1α at shear stress rate of 0.75 dyne/cm². The number of adherent cells is expressed as the mean plus or minus SEM, *indicates P less than .02. (C) The percentage of adherent cells with polarized morphology was determined. Cell polarization was inhibited by the PLC inhibitor (U73122), whereas PKC inhibition (Gö 6850) had no observable effect. Data are the mean plus or minus SEM, *indicates P less than .02. (D) PLC-dependent activation of LFA-1. Flow cytometry of T cells incubated with 10 μM U73211 (left) or 10 μM Gö 6850 (right) for 10 minutes and stained with the high-affinity extension reporter mAb KIM127 in the absence (Basal) or presence of SDF-1α. Inhibition of PLC prevented the exposure of the KIM127 neoepitope in response to SDF-1α, whereas PKC inhibition had no effect. Three to 5 independent experiments were done for each panel.