PMA stimulates the release of tie from the cell surface and the accumulation of a soluble tie fragment in the media. HUVECs were incubated in basal media with or without 10 ng/mL PMA for the indicated length of time, after which the conditioned media was collected, and the cell layer rinsed and lysed as described in Materials and Methods. Cell lysates (A) or conditioned media (B) were immunoprecipitated with anti-tiex. Immunoprecipitates were analyzed on SDS-PAGE followed by immunoblotting with anti-tie MoAb 21G6. Accumulation of a protein band at ≈100 kD can be observed in the conditioned media of PMA-treated cells starting at 15 minutes. (C) CHO, clone 37 cells were treated with PMA for the indicated times, after which, cell lysates or conditioned media supernatants (SNs) were immunoprecipitated with anti-tie MoAb 5D2. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with anti-tie MoAb 21G6. CHO clones constitutively released a significant level of soluble tie into the media, although treatment with PMA increased soluble tie levels significantly. (D) MDEC cells were treated with PMA for the indicated times before preparation of cell lysates or collection of conditioned media supernatants (SNs) for analysis by immunoprecipitation and immunoblotting as described. Molecular weight markers as indicated.