MyH9–LFA-1 association is not required for CXCL-12–induced LFA-1 activation. (A) T lymphocytes were stimulated with (filled histograms) or without (open histograms) 100 ng/ml CXCL-12 for the indicated time period. Cells were labeled with KIM127 antibody and subjected to flow cytometry. Experiments were performed in triplicate with cells from three different donors. Representative histograms showing the time course of KIM127 epitope expression are shown. (B) Human T lymphocytes adherent to ICAM-1–coated slides were stimulated with 100 nM CXCL-12. Nonadherent cells were removed, and the number of bound cells was counted. Data are expressed as mean ± SEM of three experiments. The experiment is representative of results from three independent donors. (C) Lysates obtained from CXCL-12–stimulated T lymphocytes were subjected to α_L (TS2/4 antibody) and β₂ (CBR LFA-1/2 antibody) immunoprecipitation, and then to silver staining. Mouse IgG antibody served as a negative control. (D) T lymphocytes were pretreated with 50 μM blebbistatin or DMSO and analyzed as described above (A). A representative result of three separate experiments is shown. (E) Data represent the mean instantaneous velocities of 10 representative T lymphocytes without (Control) or with 50 μM blebbistatin (Blebbistatin), respectively, in a parallel-plate flow chamber coated with CXCL-12 and ICAM-1 at a wall shear stress of 0.5 dyn/cm². T lymphocyte trajectory was monitored for 2 s before the first contact between cells and substrate (arrow) and arrest (time 0). The time interval between data points is 33 ms. All data are expressed as mean ± SEM.