Biochemical detection of ADAP in complex with talin, kindlin-3, and β3 in human and mouse platelets. (A) Shown is a primary sequence schematic of ADAP, illustrating regions known to be necessary for interaction with the indicated binding partners. (B-E) Washed human platelets were stimulated for the indicated times (or for 8 minutes if not indicated) with an activation peptide for human PAR1 (SFLLRN). Platelets were lysed with NP40 buffer containing protease inhibitors and used for immunoprecipitation (IP) (B-C) or pull-down assays (D-E), followed by Western blotting using the indicated antibodies. Unstimulated platelet lysate is shown as a positive control. For pull-down assays, lysates from human (D-E) or unstimulated SKAP2^+/+ and SKAP2^−/− murine (F) platelets were incubated with purified GST, GST-talin (D,F), or GST-kindlin-1 (E). Coomassie staining of 10% loading samples from the same gel indicated similar GST-fusion protein loading, (G) blotting of the murine platelet lysate with an antibody to SKAP2 confirmed the genotypes, and (H) blotting with an antibody recognizing human and mouse SKAP1 that confirmed SKAP1 expression in Jurkat cells, but not in human or SKAP2^−/− platelets. Results are representative of at least 3 experiments.