Hyperphosphorylated p32/p30 coimmunoprecipitated with SHP-1 and SHP-1-C from D/C cells but were not recognized by SHP-1 SH2 domains in vitro. (A) TCLs were prepared from D/V and D/C cells and were used for immunoprecipitation with antibodies specific for SHP-1 (lanes 5 and 6) or SHP-1-C (lanes 3 and 4). TCLs and the immunocomplexes were analyzed by SDS-PAGE/Western blotting with antibodies as indicated. (B) Cell lysates of D/V or D/C cells stimulated without (−) or with (+) Epo for 5 minutes were used in anti-EpoR immunoprecipitation (lanes 1 through 4) or in in vitro binding assays with a GST fusion protein of SHP-1 SH2 domains containing amino acids 1-198 of the murine SHP-1 (lanes 5 through 8). Phosphoproteins were analyzed by SDS-PAGE/Western blotting with an anti-ptyr antibody. The higher level of EpoR phosphotyrosine signaling from D/C cells (lane 8) was not reproducible and may have been caused by variations in the amount of fusion proteins. The positions of phosphoproteins and protein size markers are indicated.