Figure 7. Morphology, Stoichiometry and ProIL-1β Processing in Inflammasomes.
A. Morphology of anti-ASC immuno precipitated NLRP3 inflammasomes from uric acid crystal activated THP-1 cells analyzed by negative stain EM. Arrows denote filaments.
B. Immuno gold EM on ultra thin cryo sections from ASCFL-eGFP transfected COS-1 cells. The ASC-containing compact structure is densely decorated by gold particles (10 nm). N, nucleus; NM, nuclear membrane.
C, D. Quantification of immuno precipitated ASC-containing complex (IP) from uric acid crystal activated THP-1 cells using quantitative anti-ASC (C)and anti-caspase-1 p12 (D) Western blotting. Known amounts of recombinant His-MBP-ASC and His-GFP-caspase-1 were Western blotted to generate standard curves. The full-length caspase-1 and the cleaved p12 bands were both included in the quantification.
E. AIM2 inflammasome reconstitution in HEK293T cells to define the functional consequence of structure-based mutations in AIM2. Cells were co-transfected with plasmids encoding proIL-1β and caspase-1 (lane 1), plus ASC alone (lane 2), or WT AIM2 alone (lane 11), or ASC together with WT or indicated AIM2 mutants (lanes 3 to 10). Maturation of proIL-1βinto biologically active IL-1β was detected by Western blotting using anti-IL-1β antibody (top panel).The expression levels of HA-ASC and Flag-AIM2 were detected by Western blotting using anti-HA and anti-Flag antibodies (lower panels).
See also Figure S7.