Molecular effects of glycosylation inhibitors on P-selectin mRNA expression (A) and the proteasomal degradation of P-selectin (B) on HUVECs.

<p><b>A</b>. Tunicamycin has no effect on P-selectin mRNA levels. HUVECs were incubated with 2.5 or 5 µg/mL tunicamycin for 48 hours before 1 U/mL thrombin was added. Total RNAs were prepared and P-selectin mRNA levels were determined by real time-PCR analysis by using 18S rRNA as an internal control. The P-selectin mRNA level of untreated HUVECs was set to 1. Lanes: 1, untreated HUVECs; 2, thrombin-stimulated HUVECs; 3, HUVECs pretreated with 2.5 µg/mL tunicamycin; 4, HUVECs pretreated with 2.5 µg/mL tunicamycin and then stimulated by 1 U/mL thrombin; 5, HUVECs pretreated with 5 µg/mL tunicamycin and then stimulated by 1 U/mL thrombin. **, p<0.0001. <b>B</b>. Effects of tunicamycin on proteasomal degradation of P-selectin. HUVECs were pretreated for 1 hour with or without 1 µM MG132, treated for 48 hours with no, 2.5, or 5 µg/mL tunicamycin, and then treated with or without 1 U/mL thrombin. Cell lysates (30 µg) prepared from HUVECs were analyzed by Western blotting as described in Materials and Methods. β-Actin was used for the loading control. The ratio of the P-selectin protein level to that of β-actin was set to 1. Lanes: 1, untreated HUVECs; 2, thrombin-stimulated HUVECs; 3, HUVECs pretreated with 2.5 µg/mL tunicamycin; 4, HUVECs pretreated with MG132 and then stimulated with thrombin; 5, HUVECs pretreated with MG132, then with 5 µg/mL tunicamycin, and stimulated with thrombin. Experiments were repeated three times that gave similar results and a representative blot is shown. **, p<0.0001 <b>C</b>. Effects of glycosylation inhibitors on AKT phosphorylation. HUVECs were cultured for 48 hours in the presence or absence of tunicamycin or castanospermine at various concentrations followed by activation with 1 U/mL thrombin. Western blot analysis was performed to determine the phosphorylation status of AKT (Ser473 and Thr308). β-Actin was the loading control. <b>D</b>. AKT inhibitor suppresses CD62P expression on HUVECs. Flow cytometric analysis of P-selectin (CD62P) expression on HUVECs treated with the AKT inhibitor LY-294002 for 1 hour and subsequently stimulated with 1 U/mL thrombin. CD59-FITC was used as the positive marker for ECs. Flow cytometry analyses were repeated three times and a representative result was shown.