Fig. 6. Tyrosines in the ⁹⁸³NPAYY⁹⁸⁷ motif are important sites of phosphorylation. (A) Wild-type and YY-FF mutant of FLAG-tagged SHIP2 were transiently expressed in HeLa cells and anti-FLAG immunoprecipitates from adherent control (-), adherent EGF-treated (50 ng/ml for 5 minutes; E), detached (D) or re-plated on collagen I (6 µg/cm² for 60 minutes; C.I) were blotted with anti-phosphotyrosine (-PY) or anti-FLAG (M2) antibodies. (B) Wild-type and three Y-F (497, 747 and 1135) point mutant SHIP2 proteins expressed in HeLa cells were analyzed as above. (C) In vitro phosphorylation reactions were carried out using recombinant Src and immunopurified wild-type or YY-FF mutant SHIP2. Samples were blotted with anti-phosphotyrosine and anti-FLAG (M2) antibodies. The arrows point to tyrosine-phosphorylated SHIP2.