Figure 1.

Purification of CEACAM1_cyt -associated proteins from granulocyte extracts. A: SDS-PAGE and silver staining of eluates from immobilized CEACAM domains. Extracts from granulocytes were subjected to purification on adjusted amounts of immobilized in vitro tyrosine phosphorylated CEACAM1_cyt (lane 1), unphosphorylated CEACAM1_cyt (lane 2), in vitro tyrosine phosphorylated CEACAM3_cyt (lane 3), and unphosphorylated CEACAM3_cyt (lane 4). Positions of the purified proteins mentioned in the text and apparent molecular masses are indicated on the left (in kilodaltons). B: Coprecipitation of integrin β₃ and CEACAM1. Extracts from granulocytes (lanes 1 and 3) and HT29 cells (lanes 2 and 4) were subjected to immunoprecipitation with CEACAM1 mAb 12-140-4 (lanes 1 and 2) or anti-integrin β₃ mAb (lanes 3 and 4). Bound proteins were resolved by 7.5% SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-integrin β₃ mAb (lanes 1 and 2) or anti-CEACAM1 mAb 4D1/C2 (lanes 3 and 4). The positions of integrin β₃ and CEACAM1 are indicated on the left or right. C: Coprecipitation of tyrosine phosphorylated wild-type and mutant CEACAM1_cyt with anti-integrin β₃ mAb. Following in vitro tyrosinephosphorylation, the radioactively labeled phosphoproteins (wild-type CEACAM1_cyt (lane 1), Y515F CEACAM1_cyt (lane 2), and Y488F CEACAM1_cyt (lane 3)) and Protein G PLUS/Protein A-agarose were incubated with precleared granulocyte extracts containing integrin β₃ mAb at 4°C for 2 hours. After immunoprecipitation, precipitates were resolved with 17.5% SDS-PAGE followed by autoradiography. Positions of labeled phosphoproteins areindicated on the left or right.