Pulse-chase analysis of VWF and AgII processing and secretion in HUVECs. (A) Phosphor-Imager data of VWF (top) and AgII (bottom) immunoprecipitates from cell lysates (Cells) and chase media (Medium) of HUVECs metabolically labeled with [³⁵S]-cysteine/methionine for 30 minutes and subsequently chased for the times indicated. Immunoprecipitates were separated by SDS-PAGE on 6% polyacrylamide gels and exposed to a PhosphorImager plate for 7 days (see “Immunoprecipitation”). (B) PhosphorImager data of VWF immunoprecipitates from cell lysates (C) and chase medium (M) of HUVECs metabolically labeled as in panel A and chased for 24 hours. Immunoprecipitates were treated with 25 mM Endo H (+) or buffer alone (−). (C) Quantification of pulse-chase experiments such as that shown in panel A. The left panel shows the time course for the appearance of mature VWF (▴) and AgII (●) and the disappearance of proVWF (■) in cell lysates. The right panel shows the corresponding time course for the appearance of these proteins in the media. The radiolabeled bands were quantified as described in “Immunoprecipitation.” The value for each time point was calculated from duplicate observations made in each of 2 independent experiments. Within each experiment the mean value of duplicates at each time point was calculated and expressed as a percentage of the maximum mean observation. The plotted data represent the means (± range) of these percentages over the 2 independent experiments. The experiment was repeated on 3 further occasions with slightly different time points but yielding equivalent results.