Brefeldin A inhibits constitutive secretion of VWF but has a modest effect on the nonstimulated release of VWF from HUVECs. (A) PhosphorImager data of VWF (left) and AgII (right) immunoprecipitated from cell lysates (C) and medium (M) of HUVECs metabolically labeled with [³⁵S]-cysteine/methionine for 30 minutes and chased in the presence (BFA) or absence (Control) of 5 μM BFA for 6 hours. (B,C) A time course of appearance of VWF immunoreactivity, determined by ELISA, in fresh growth medium (□) and fresh growth medium containing 5 μM BFA (▴) from HUVECs (B) and CHO cells stably expressing VWF (C). The amount of VWF immunoreactivity secreted is expressed as IU/mL after calibration against a known standard (see “Measurement of VWF by enzyme-linked immunosorbent assay”). In each case data points represent the means (± range) of duplicate observations in a representative experiment. Experiments in CHO were repeated on a further 2 occasions with similar results. The experiment in HUVECs was repeated on many occasions (n = 17) with variable results, see discussion in “BFA blocks secretion of newly synthesized VWF but does not abolish spontaneous release of VWF.”