SHIP-2 associates with filamin independently of actin. (A) Schematic of the calpain-mediated cleavage profile of filamin A. The empty box represents the actin-binding domain (ABD) and the repeats 1-24 of filamin are shown as ovals. Gray and black filled ovals represent GPIb and SHIP-2 binding sites, respectively. The calpain-cleavage sites on intact filamin are shown. Calpain-mediated cleavage of filamin results in the production of 100-kDa and 90-kDa fragments, which retain the GPIb and partial SHIP-2 binding sites, and a 190-kDa fragment, which contains the actin-binding domain but lacks the SHIP-2 and GPIb binding site. (B) The Triton X-100–soluble fraction of platelets isolated in lysis buffer containing EGTA (lanes 1, 4), in the absence of EGTA (lanes 2, 5), or in the presence of DNase I without EGTA (lanes 3, 6) was immunoprecipitated with either preimmune (Pre I) sera or SHIP-2 affinity-purified antibodies and immunoblotted with filamin antibodies (top), actin antibodies (bottom), or SHIP-2 antibodies as shown. Arrows indicate the migration of intact filamin and calpain-cleaved filamin fragments.