Angiopoietin 1

Previously described procedures were used for the ELISAs with some modifications (32 (link), 33 (link)). EIA/RIA plates (Corning Incorporated, Corning, NY) were coated with 100 μL of purified recombinant protein at a concentration of 5 μg/mL in coating buffer (0.05% sodium azide containing PBS) overnight at 4°C. Angiopoietin-1 and angiopoietin-2 were from R&D, tissue-type plasminogen activator was from Abnova, and recombinant ML-IAP and NY-ESO-1 were prepared in house. The plates were washed with PBST (0.05% Tween-20 containing PBS) and blocked for two hours at room temperature with 200 μL/well blocking solution (PBST, 2% nonfat milk, 0.05% sodium azide). After the plates were again washed, longitudinal sera samples were added at a final dilution of 1:500 in blocking solution (100 μL/well) and incubated at 4°C overnight. After several further washes, the plates were incubated with 100 μL/well of a 1:2000 diluted alkaline phosphatase–conjugated goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for one hour at room temperature. Finally, the plates were washed again, incubated with 100 μL/well of the PNPP substrate (Sigma, St. Louis, MO) for 25 minutes at room temperature, and then the OD (405 nm) was determined (Spectramax 190 Microplate Reader; Molecular Devices, Sunnyvale, CA).

EBM-2 basal medium served as vehicle for drug treatments, and as the vehicle control. Angiopoietin-1, CD14 and LPS binding protein were purchased from R&D Systems (Minneapolis, MN). Thrombin was purchased from EMD Millipore (Billerica, MA). LPS serotype O111:B4 was purchased from Sigma-Aldrich (St. Louis, MO). Y-27632 was purchased from EMD Biosciences (LaJolla, CA).