The multiple forms of SHIP can be generated in vitro by digestion with calpain. (A) Lysates from IL-3–stimulated DA-ER cells expressing HA-SHIP were immunoprecipitated with anti-N (lane 1) or anti-C (next 3 lanes), and the latter was subjected to digestion with 10 mU of pure rabbit m-calpain for the times indicated. The control sample (lane 2) was incubated for 15 minutes in the absence of m-calpain. The samples were then subjected to anti-HA Western analysis. (B) Lysates, prepared in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of the standard protease inhibitors (see the Materials and Methods) from IL-3–stimulated DA-ER cells expressing HA-SHIP, were immunoprecipitated with anti-C (lanes 1, 2, and 3) or anti-N (lane 4) and the lane 3 sample was digested with 10 mU of pure rabbit m-calpain for 4 hours. The samples were then subjected to anti-HA Western analysis.