Blockage of receptor internalization prevents the thrombin-induced down-regulation of the serine-phosphorylated endoglin by TβRII in ECs. (A, top) HAECs were left untreated (control) or pretreated with okadaic acid (OA; 100 nM) for 30 minutes, then stimulated with thrombin (3 U/mL) for the indicated time periods. (Bottom) Optical density. ▪ represents control (no thrombin stimulation); ▦, thrombin 30 min; □ thrombin 1 h. Error bars indicate standard deviation. (B) HAECs were left untreated (control) or pretreated with pervanadate (PV; 50 μM) for 30 minutes or phenylarsine oxide (PAO; 1 μM) for 3 hours, then stimulated with thrombin for 30 minutes. (C, top) Control cells (+K) or HAECs depleted of potassium (–K) were stimulated with thrombin (3 U/mL) for the indicated time periods. (Bottom) Optical density. Shading indicates same treatment types as in panel A. Error bars indicate standard deviation. (D) HUVECs were left untreated (control) or pretreated with 0.45 M sucrose for 10 minutes then stimulated with thrombin for the indicated time periods. Lysates were subjected to immunoblotting with the antibody against the serine-phosphorylated endoglin as described in Figures 1 and 2. The same blot was stripped and reprobed with an endoglin antibody (SN6h) to show the equal loading. Results shown are representative immunoblots and densitometric analyses of 3 independent experiments.