Lad is required for chemokine-induced T-cell migration. (A) Suppression of endogenous Lad expression by siRNA inhibits chemokine-dependent T-cell migration. Jurkat T cell clones that stably express CCR5 were transfected with pSUPER plasmid expressing control siA or siA. Subsequently, 5 × 105 cells were loaded into the upper part of a Transwell chamber inserted into medium containing 25 ng/mL or 50 ng/mL SDF-1α or RANTES. After incubation for 2 hours, cells that had migrated into the bottom chamber were counted. The experiments were performed 3 times in duplicate, and the results were expressed as mean ± SD. Error bars represent the standard deviation. As a control, protein level of endogenous Lad was analyzed by Western blotting with antiLad antibody (inset). (B) Jurkat T-cell clones that stably express Lad (Lad) or the SH2 domain of Lad (Lad-SH2) were subjected to the chemotaxis assay described in panel A. Two independent clones were tested for each transfectant.
