Fig. 3. The SHP-2-interacting p120 tyrosyl-phosphorylated protein is SHPS-1. (A) (Left) Proliferating C2C12 myoblasts cultured in GM were lysed and immunoprecipitated with either pre-immune (PI) or anti-SHP-2 antibodies. Immune complexes were resolved by SDS-PAGE and immunoblotted with anti-SHPS-1 (epitope directed to the conserved cytoplasmic domain of SIRP family members). (Right) C2C12 myoblast lysates prepared as described above were subjected to immunoprecipitation with anti-SHPS-1/MFR (10C4) antibodies, which recognize the V1 hypervariable extracellular region of SHPS-1. Immune complexes were immunoblotted with anti-SHPS-1 antibodies. (Bottom) The above immunoblots were reprobed with anti-SHP-2 antibodies. (B) Lysates were prepared from C2C12 myoblasts cultured in either GM or DM for the indicated times and were immunoblotted with anti-SHPS-1 antibodies. (C,D) Tyrosyl phosphorylation of SHPS-1 and association with SHP-2 during C2C12 myogenesis. Lysates prepared from C2C12 myoblasts cultured in either GM or DM for the indicated times were subjected to immunoprecipitation with either anti-SHPS-1 antibodies, followed by pTyr immunoblotting (C) or immunoprecipitation with anti-SHP-2 antibodies followed by anti-SHPS-1 immunoblotting (D). The lower panel in C represents an equal fraction of the lysates from the experiment above, which was immunoprecipitated for SHPS-1 and immunoblotted with anti-SHPS-1 antibodies as a control. The lower panel in D is a reprobe of the above experiment with anti-SHP-2 antibodies. The positions of molecular mass markers are shown.
