LFA-1 is associated with Lck and ZAP-70 in a signaling complex that is activated in response to LFA-1 binding to ICAM-1. (A) Total T-lymphoblast lysate (TL); LFA-1 immunoprecipitates from T lymphoblasts in suspension (IP) blotted for the presence of LFA-1, Lck, ZAP-70, Fyn, and α-tubulin; n = 4. (B) Total primary T-lymphocyte lysate (TL); LFA-1 immunoprecipitates from primary T lymphocytes in suspension (IP) blotted for the presence of LFA-1, Lck, ZAP-70, Fyn, and α-tubulin; n = 3. (C) Lysates of T lymphoblasts either in suspension (Susp) or treated with pan–LFA-1 mAb YTH81.5 and blotted for phospho-Lck (Y394), phospho-ZAP-70 (Y319), and α-tubulin; n = 4. Black vertical lines indicate images taken from separate tracks on the blots. (D) T lymphoblasts were incubated with either ICAM-1– or BSA-coated microspheres for 15 minutes at 37°C. Typical wide field images are shown of the interaction of the T cells (dark images) with the coated microspheres (white spheres). (E) T blasts were incubated for 30 minutes with the microspheres, then lysates were made and blotted for total LFA-1 (control), phospho-Lck (Y394), and phospho-ZAP-70 (Y319). Black vertical lines indicate images taken from separate exposures of the blots. (F) T lymphoblasts either untreated or treated with 40μM PP2 or 10μM piceatannol were incubated on ICAM-1–coated plates. Lysates were probed for phospho-Lck (Y394), total Lck, phospho-ZAP-70 (Y319), total ZAP-70, and α-tubulin by Western blotting; n = 3.