Effects of cell treatment with TAT-CRIB.

(A) TAT-CRIB does not inhibit SDF-1–induced actin polymerization. After a 2-hour incubation in medium containing 300 μg/mL TAT-CRIB or not (control), CEM cells were stimulated with SDF-1 (300 ng/mL) for 30 seconds at 37°C. Cells were then fixed in paraformaldehyde, permeabilized, and incubated with fluorescein isothiocynate–labeled phalloidin. Analysis of fluorescein isothiocynate intensity on a FACSort (Becton Dickinson) showed that the baseline content of F-actin (dotted line) and its increase following SDF-1 stimulation (plain line) are not altered by TAT-CRIB. (B) Effects of TAT-CRIB on PAK-2 and -4 activities. Cells were preincubated with 300 μg/mL TAT-CRIB (+) or not (−) for 2 hours. Cells were then left unstimulated or were stimulated with 500 ng/mL SDF-1 for 5 (PAK-2) or 20 minutes (PAK-4) and lysed. The lysates were immunoprecipitated with anti–PAK-2 or anti–PAK-4, and PAK activity was determined by in vitro kinase assay. Data shown represent mean + SE of 4 (PAK-2) or 3 (PAK-4) independent experiments. Representative autoradiograms are shown in insets.