Release of mature caspase-1 is ASC dependent but extracellular caspase-1 activity does not co-precipitate with ASC or predominate in microvesicles.

<p>A) THP1 (2x10⁷ cells/ml) cells stably transduced with shRNA for ASC (siASC) and EGFP (siEGFP control) were left untreated or stimulated with LPS/ATP (L/A) for 1h and caspase-1 activity from the supernatants was measured. B) Lysates and supernatants from <i>(A)</i> were probed for caspase-1, ASC, and β-actin by immunoblot. C) Supernatants of LPS/ATP treated THP1 cells (2x10⁷ cells/ml) were analyzed for WEHD-afc activity after immunodepletion with either monoclonal anti-ASC antibody (α-ASC) or control mouse IgG1A (IgG) (top panel). Immunoblots for caspase-1 and ASC confirm effective ASC depletion (bottom panel), immunoprecipitate beads <b>(B)</b> and residual supernatant <b>(Sup)</b>. Co-immunoprecipitation of caspase-1 and immunoprecipitation of ASC are shown below. Original supernatant (-), immunoprecipitate beads (B) and residual supernatant (Sup). * indicates IgG light chain. D) Supernatants of LPS/ATP treated THP1 cells (2x10⁷ cells/ml) were subjected to microvesicle purification by differential centrifugation. Sup1 (control, 16,000g supernatant), Sup2 (100,000g supernatant), MV (100,000g pellet). Caspase-1 activity was measured in the MV-enriched (Sup1), MV-depleted (Sup2), and MV pellet. Fractions were run on immunoblot and LAMP-1 was used as a marker to confirm microvesicle enrichment [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142203#pone.0142203.ref037" target="_blank">37</a>]. Representative gel and data expressed as mean ± SEM from n = 3 independent experiments (A, B, C and D).