Thrombin induces a PKC-ζ–dependent internalization of endoglin and TβRII in ECs. (A-B) HUVECs grown on glass coverslips were left untreated (control) or pretreated with myristoylated PKC-β or PKC-ζ peptide inhibitors, then stimulated for 30 minutes with thrombin (3 U/mL), washed twice, and fixed. Endoglin (A) and TβRII (B) were viewed (10 × 100) by staining the fixed cells with antibodies against L-endoglin (SN6h) or TβRII (C-16). Arrows indicate the internalized endoglin or TβRII. (C) HUVECs were left untreated (–) or pretreated for 30 minutes with GF109203X (GFX; 15 μM), rottlerin (Rott; 10 μM), Gö6976 (Gö; 300 nM), or LY294002 (LY; 20 μM), then stimulated with thrombin (3 U/mL) for 30 minutes. (D) HUVECs were stimulated with thrombin (3 U/mL) for 30 minutes or phorbol 12-myristate 13-acetate (PMA; 100 nM) for 10 or 30 minutes. Lysates were subjected to immunoblotting with the antibody against the serine-phosphorylated endoglin as described in Figures 1 and 2. (E) HUVECs were stimulated with thrombin (3 U/mL) for the indicated time periods. Lysates were immunoblotted with an antibody against phospho–PKC-ζ/λ (Thr-410/403). (F) HUVECs were left untreated (control) or pretreated with a myristoylated PKC-ζ peptide inhibitor for 30 minutes, then stimulated with thrombin (3 U/mL) for 30 minutes. The serine phosphorylation of endoglin was determined as described in Figures 1 and 2. Results shown are representative of 3 independent experiments.