ADAP is involved in outside-in signals by LFA-1, and ADAP ligation induces actin cloud formation. (A) Phosphorylation of ADAP upon LFA-1 stimulation. Jurkat cells were stimulated for 10 minutes with anti–LFA-1 followed by anti–mouse Ig (+) or left unstimulated (−), and cell lysates were immunoprecipitated with anti-ADAP Ab and blotted with anti-PY Ab. (B) Association of LFA-1 with ADAP upon LFA-1 stimulation. Jurkat cells were stimulated for 10 minutes with anti–LFA-1 or left unstimulated (None), and the cell lysates were immunoprecipitated with anti–LFA-1β and blotted with anti-ADAP or anti–LFA-1β. The amounts of ADAP in total lysates were equivalent (bottom blot). (C) Schematic structure of CD8/ADAP constructs. (D) Direct ligation of ADAP induces actin cloud formation. CD8/ADAP- or CD8-expressing Jurkat cells were stimulated on coverslips coated with anti-CD8 for 10 minutes, fixed, and stained with phalloidin–Alexa 568. Most of the T cells (> 80%) formed actin clouds (top panels). The surface expression of CD8 was analyzed by flow cytometry using FACSCalibur (bottom panels). (E) ADAP activation induces the accumulation of tyrosine-phosphorylated proteins in the actin cloud. Jurkat cells, as in panel D, were stained with phalloidin–Alexa 488 and anti-PY–biotin, followed by avidin-Cy3. The white arrow indicates the representative actin cloud in panels D and E. More than 100 cells were analyzed for panels D and E.