The ability of SHIP to bind Shc does not affect MAPK phosphorylation.

(A) SHIP^+/+ and SHIP^−/− BMMCs (4 × 10⁷) as well as SHIP^−/− BMMCs expressing the indicated SHIP constructs were treated with or without 400 ng/mL SF for 2 minutes and NP40 cell lysates subjected to immunoprecipitation with anti-Shc antibodies and Western analysis with 4G10. The lower panel is a reprobe with anti-Shc antibodies to show equal loading. (B) At the same time as panel A was carried out, 1 × 10⁶ cells/sample were lysed with SDS-sample buffer and subjected directly to Western analysis with anti-phosphoMAPK antibodies. The lower panel shows a reprobe of this blot with anti-MAPK antibodies to demonstrate equal loading. (C) SHIP^−/−BMMCs expressing the indicated constructs were treated with or without 400 ng/mL SF for 2 minutes and the NP40 cell lysates subjected to immunoprecipitation with anti-Shc antibodies and Western analysis with anti-HA antibodies (top panel). The bottom panel shows a reprobing of this blot with anti-Shc antibodies to demonstrate equal loading. The NP40 cell lysates used in panel C were from the 2-minute time point in Figure 4 and thus contained equivalent levels of SHIP (Figure4B).