Binding properties and subcellular location of the various SHIP proteins. (A) Cell lysates from HA-SHIP–expressing DA-ER cells, treated with IL-3 for 5 minutes at 37°C, were either subjected directly to SDS-PAGE (right panel) or were incubated with beads containing the C-terminal SH3 domain of Grb2 or the PTB domain of Shc for 1 hour at 4°C, or immunoprecipitated with anti-Shc antibodies. The beads were then washed 3 times with PSB containing 0.5% NP40 and boiled in SDS-sample buffer and all of the samples were subjected to anti-HA Western analysis. (B) DA-ER cells expressing HA-SHIP were stimulated with IL-3 for the indicated times and fractionated into TX-100–soluble (Tx sol), TX-100–insoluble (Tx insol), and submembranous cytoskeletal (SM Cyto) fractions and subjected to anti-N immunoprecipitation and anti-HA Western analysis. (C) (Left panel) Total cell lysates from DA-ER and murine bone marrow cells were subjected to anti-N+anti-M Western analysis. (Right panel) Murine bone marrow cells (2 × 10⁷ cells) were lysed in 100 μL 2% TX-100 in PSB for 10 minutes at 4°C and centrifuged at 10,000g for 10 minutes. The TX-100–insoluble fraction was extracted from the pellet with 100 μL 1% TX-100, 0.3% deoxycholate, and 1 mol/L NaCl in PSB for 30 minutes at 4°C and, after centrifuging at 10,000g for 10 minutes, it (the supernatant) was subjected to anti-N+anti-M Western analysis.