Inhibition of soluble tie release by downregulation of PKC. HUVECs were incubated with 16 nmol/L (10 ng/mL) PMA for 1 hour, 1 μmol/L PMA for 24 hours, or both, as indicated, before harvesting of conditioned media supernatants and preparation of cell lysates. Lysates and supernatants were immunoprecipitated with anti-tie MoAbs, and analyzed by SDS-PAGE followed by immunoblotting as described in Materials and Methods.