Conversion of HUVEC-bound PK to kallikrein. HUVECs were incubated with buffer (A) or with 20 nmol/L HK (B) for 1 hour at 37°C. Unbound HK was removed and 20 nmol/L biotin-PK was added for 1 to 120 minutes at 37°C. After washing, the cells were solubilized by adding electrophoresis sample buffer containing 2% β-mercaptoethanol. The proteins were boiled and then separated on 10% SDS-PAGE and electroblotted onto nitrocellulose, and biotin-PK was detected by adding streptavidin-horseradish peroxidase. Photographs of ligand blots on nitrocellulose are shown. The numbers on each side of the photographs are molecular mass standards in kilodaltons. The numbers between the photographs show the time of incubation with PK. CN represents soluble biotin-PK starting material and XIIf represents βFXIIa-cleaved biotin-PK analyzed by SDS-PAGE in the absence of cells.