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Fig. 6. Tyrosine-phosphorylated hSpry2 is associated with SRC. (A) hSpry2 was associated with SRC. hSpry2WT was transfected into SYF cells alone or SYF cells stably expressing wtSRC (SYF/wtSrc), kinase active SRC (SYF/Src Y527F) or a kinase dead SRC (SYF/Src KD). Cells were subsequently stimulated with FGF2 (30 minutes). SRC was immunoprecipitated with a monoclonal anti-SRCMab327 antibody and immunoblotted with anti-SRC (SRC2) antibody and hSpry2 association was examined using anti-hSpry2 antibody. Relative expression and phosphorylation of hSpry2 was confirmed with either anti-hSpry2 antibody or anti-phosphotyrosine antibodies. (B) Preferential pY55-mediated association of SRC with hSpry2. Peptides derived from hSpry2 that included Y55 in both its phosphorylated (lanes 1) and non-phosphorylated (lanes 2) form were used to isolate potential binding partners. Peptide-associated proteins were resolved on SDS-PAGE gel, and the presence of SFKs revealed utilizing anti-phosphotyrosine antibodies and anti-SFK (SRC2) antibody.





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