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. 2009 Oct 30;284(52):36062–36076. doi: 10.1074/jbc.M109.064923

FIGURE 6.

FIGURE 6.

SHIP2 monoubiquitination is regulated by EGF stimulation. A, COS-7 cells were transiently transfected with FLAG-tagged ubiquitin and His-tagged SHIP2 or an empty vector, serum-starved for 16 h, and stimulated with 50 ng/ml EGF for 5, 30, and 60 min. Equal amounts of cell lysates were immunoprecipitated with an anti-FLAG antibody and immunoblotted with anti-SHIP2 antibodies. Expression of SHIP2 was confirmed by immunoblotting of the cell lysates with anti-SHIP2 antibodies. B and C, COS-7 cells were transfected with His-tagged SHIP2 (B) or with HA-tagged c-Cbl (C), serum-starved for 16 h, and stimulated with 50 ng/ml EGF at 5, 15, and 30 min. Equal amounts of cell lysates were immunoprecipitated (IP) with an anti-His antibody, and immunoprecipitates were separated by SDS-PAGE and immunoblotted (IB) with anti-4G10 (anti-phosphotyrosine) or anti-His antibodies (B) or with anti-Cbl or anti-His antibodies (C).