Figure 5.

Degradation of P-selectin is prevented by a proteinase inhibitor cocktail. NRK cells expressing native P-selectin were labeled with biotin and then either harvested (0 chase) or recultured for 3 h in the presence of a proteinase inhibitor cocktail (containing AEBSF, aprotinin, bestatin, E64, leupeptin, and pepstatin A) diluted 1:1000 (1× PIC) or 1:500 (2× PIC) or in the presence of DMSO alone diluted 1:500 (DMSO). P-selectin was immunoprecipitated from detergent lysates of the cells, and the remaining biotin was measured as described for the turnover experiment in Figure 4. Numbers represent the average of duplicate samples, expressed as the percentage of P-selectin remaining, normalized to 0 chase. The proteinase inhibitor cocktail potently inhibited degradation of P-selectin.