Cycloheximide inhibits basal secretion of VWF. (A) HUVECs were incubated overnight (∼16 hours) with full-growth medium alone (□, ▵) or growth medium containing 5 μM cycloheximide (●, ▴). Medium was replaced with fresh full-growth medium with no additions (Control; □) or supplemented with 30 μM histamine (▵), 5 μM cycloheximide (CHX; ●), or both histamine and cycloheximide (▴) and secretion of VWF immunoreactivity into the media during a 5-hour period was measured by ELISA. Data points represent the means (± range) of duplicate observations. (B) HUVECs were metabolically labeled with [³⁵S]-cysteine/methionine for 30 minutes, chased for 5 hours in fresh full-growth medium before a further 24-hour incubation in growth medium with (CHX) or without (Control) 5 μM cycloheximide. Radiolabeled VWF secreted into fresh growth medium alone (Control) or fresh growth medium supplemented with 5 μM cycloheximide (CHX) during a further 5-hour period was immunoprecipitated, separated by SDS-PAGE, and quantified (Med), along with the VWF remaining in the cell lysates (Cells) at the end of the experiment. Representative PhosphorImager data are shown. Mature VWF is indicated by ▶. (C) Amount of VWF recovered from cells and medium expressed relative to Control values. Values represent means (± SD), n = 9.