FXII cleavage by PKa and PK. Western blots of time courses of cleavage of FXII or FXII-S544A (200 nM) by (A) PKa (50 nM). At indicated time points, samples were removed into nonreducing sample buffer, size fractionated by SDS-PAGE, and analyzed by western blotting using monoclonal anti-FXIIa IgG D06. Positions of standards for αFXIIa and βFXIIa are indicated at the right of each image. (B) Western blots of time courses of cleavage of FXII-S544A (200 nM) by recombinant PK (PK-WT, PK-R371A, or PK-S544A) in the presence of vehicle (top row), poly-P (70 μM, second row), or poly-P and C1INH (750 nM, third row). Blots were processed as in panel A. Positions of a standard for αFXIIa are indicated at the right of each image. The fourth and fifth rows are western blots for PK, using the same samples for reactions with vehicle and poly-P used in the first 3 rows. Positions of standards for PK, the heavy (HC) and light (LC) chains of PKa, and a heavy-chain fragment from β-kallikrein are indicated at the right of each image. (C) Western blots of time courses of cleavage of FXII-S544A (200 nM) in the presence (+) or absence (−) of PK-R371A, with human genomic DNA (25 μg/mL), 15% (vol/vol) silica containing PTT-A reagent (silica), or DXS (1 μg/mL). Blots were processed as in panel A. Positions of a standard for αFXIIa are indicated at the right of each image.