Comparison of human HK and mouse HK in their interaction with LPS. (A) BODIPY FL–LPS at 50 ng/ml was suspended in 100 µl PBS. After the addition of human HKa or mouse HKa at the indicated concentrations, the real-time change in BODIPY FL–LPS fluorescence upon transition from the aggregated LPS state was recorded in 20-s intervals. Data are representative of two independent experiments. (B) Raw 264.7 cells were incubated with, or without, human HKa or mouse HKa (μg/ml) in the presence or absence of 0.03 ng/ml LPS at 37°C for 6 h. The concentration of TNF in the culture supernatants was determined using ELISA. Statistics were analyzed using an ANOVA. *, P < 0.05; **, P < 0.01. Data are representative of two independent experiments and expressed as mean ± SEM. (C) 1 µl human or mouse plasma was solubilized in SDS sample buffer and separated by SDS-PAGE. The samples were analyzed by immunoblotting using a polyclonal anti–HK-HC antibody (left) or an anti-DHG15 antibody (right). (D) Quantification of human SCHK and mouse SCHK in plasma by immunoblotting. 1 µl kininogen-deficient human plasma supplemented with 0, 5, 10, 20, 40, 80, or 160 ng human SCHK and 1 µl fresh human plasma were electrophoresed and immunoblotted using an anti-DHG15 antibody (top). Similarly, 1 µl kininogen-deficient mouse plasma supplemented with 0, 5, 10, 20, 40, 80, or 160 ng of mouse SCHK and 1 µl fresh WT mouse plasma were electrophoresed and immunoblotted using an anti-DHG15 antibody (bottom). The intensity of bands in the immunoblots were analyzed as described in Materials and methods. A standard curve for protein concentration was created by plotting intensity as a function of protein quantity. The quantification was performed using three samples of human plasma and four samples of mouse plasma. Data are representative of two independent experiments. RFU, relative fluorescence units.