A1PI Interacts with Caspase-1 in Monocyte Cytosol.

<p>A) Cytosolic preparations of THP-1 cells activated 18 hours with PMA were applied to a Superdex S-200 column. Fractions 6 through 24 were analyzed by SDS-PAGE and immunoblot with antibodies directed to A1PI (top panel), caspase-1 (middle panel), and ASC (bottom panel). B) THP-1 cells activated overnight with PMA were pulsed for 1 hour with 20 µM nigericin before hypotonic lysis. Immunoprecipitation was performed using an irrelevant control IgG or A1PI-directed antibodies. Precipitated proteins were analyzed by immunoblot with anti-A1PI (top panel) or anti-caspase-1 (lower panel). C) Graded amounts of plasma derived A1PI, BSA control, or small molecule caspase-1 inhibitor, were coincubated with recombinant active caspase-1 prior to addition of a fluorescent peptide substrate. Reactions were incubated at 37 °C, and fluorescence over time in duplicate wells (mean and STD) were measured by platereader. D) Gelatin control protein or plasma derived A1PI were conjugated to agarose beads, then incubated with recombinant caspase-1. After extensive washing, proteins were eluted with SDS-PAGE buffer and analyzed by immunoblot for bands reactive with a caspase-1 directed polyclonal antibody.