Release of soluble tie from HUVECs is mediated through PKC activation. HUVECs were incubated with basal media alone (-PMA), 10 ng/mL PMA (+ PMA), 10 ng/mL 4αPMA (4αPMA) or 25 μmol/L HA 1004 (HA 1004) for 1 hour, or preincubated for 15 minutes with 100 nmol/L staurosporin (Staurosporin) or 200 nmol/L GF 109203X (GF109203x) before the addition of PMA for 1 hour. Preparation of cell lysates was performed as described in Materials and Methods. Samples were analyzed by immunoprecipitation using anti-tie MoAbs, SDS-PAGE, and immunoblotting as previously described.