Figure 2.

Analysis of AAT elastase complex formation in culture supernatants. PBMCs were incubated with 0.5 mg/mL plasma-derived AAT or recombinant AAT. Some cells were cultured with a combination of AAT proteins together with 10 ng/mL LPS and 12 µg/mL spike. After 6 h of incubation at 37 °C, cultured supernatants were harvested. The supernatants were further incubated with PBS or porcine pancreas elastase in a molar ration of 2:1 (AAT/elastase) for 30 min at room temperature. The complex formation was stopped by addition of SDS sample buffer and heating at 90 °C for 2 min and analyzed by Western blotting. AAT and AAT-containing complexes were targeted with rabbit polyclonal anti-AAT antibody and anti-rabbit-horseradish peroxidase and visualized with ECL Western blotting substrate. The figure shows one representative blot out of two independent experiments.