Fig. 5.

LOK depletion results in alteration of migration and polarization, but not in adhesion. (A) Splenic lymphocytes from LOK KO mice and littermate controls were analyzed for transmigration to SDF-1. (B) Splenic lymphocytes from LOK KO mice and littermate controls were analyzed for adhesion to VCAM-1 or ICAM-1. Assays were performed in the presence or absence of 1 mM MnCl₂, which promotes conversion of integrins into active conformation. (C) Splenic lymphocytes from LOK KO mice and littermate controls were analyzed for cpERM phosphorylation under 4 standard conditions: R, resting, no treatment; SDF-1, 1 min after 100 ng/mL SDF-1 to assess physiological dephosphorylation; STA, 5 min after high concentration (500 nM) staurosporine to assess maximal dephosphorylation; CA, 5 min after 50 μM phosphatase inhibitor calyculin A to maximize phosphorylation; cpERM was assessed both by WB (Upper subpanel) and flow cytometry (Lower subpanel). (D) cpERM levels were measured for splenic lymphocytes from LOK KO mice and littermate controls before and after various times of stimulation with SDF-1. (E) Cells as in panel D, but stained with phalloidin to detect F-actin and scored blind for polarization. (F) Cells as in panel E, but analyzed for increase in F-actin by flow cytometry after 15 s of stimulation.