PMA induces cleavage of the tie receptor into an N-terminal 100 kD soluble fragment and a C-terminal 40 kD cell-associated fragment. Cell lysates prepared from untreated HUVECs (A) or CHO, clone 37 cells (B) were immunoprecipitated with antibodies recognizing either the N-terminus (MoAb 5D2) or the C-terminus (SC Ab) of the tie receptor and analyzed by SDS-PAGE on a 6% (A) or 8% (B) gel. Conditioned media supernatants were prepared from HUVECs (A) or CHO, clone 37 cells (B) that had been incubated with 10 ng/mL PMA for 1 hour, followed by immunoprecipitation with Abs recognizing either the N- or C-terminal of tie and analysis on SDS-PAGE. For samples labeled N-term + sol. tie, the N-terminal Ab was preincubated with 30 μg of recombinant soluble tie before immunoprecipitations were performed. Arrows, full-length cell-associated tie; arrowheads, soluble tie. (C) HUVECs were treated in the presence or absence of 10 ng/mL PMA, as indicated, for 1 hour followed by the preparation of cell lysates. Immunoprecipitations were performed with polyclonal Abs specific for the C-terminus (SC Ab) or N-terminus (anti-tiex) of tie followed by analysis on an 8% gel and immunoblotting with the SC Ab. To show specificity, in lanes labeled C-term pep +, the SC Ab was preincubated with the C-terminus tie peptide (amino acids 1121-1138) before immunoprecipitation.