Figure 4. Ionizing radiation induces the formation AIM2 specks in the nucleus.

Primary macrophages from AIM2-Flag mice (A, B, C, D) or AIM2-Flag/ASC-HA double knock-in mice (E) were left un-irradiated or exposed to 80 Gy ionizing radiation. For immunofluorescence microscopy, cells were fixed at 4 hours (A, B, C) or at indicated time points (E) after radiation. AIM2 was stained with anti-Flag antibody (red in A; green in B, C, E), and co-stained with nuclear envelope protein NUP98 (red, B) or gamma-H2AX (red, C) or ASC (using anti-HA antibody, red, E). Cell nuclei were visualized by DAPI (blue) in A and E. Co-localization of AIM2-Flag specks and gamma-H2AX foci was indicated by white arrowheads in C. Scale bar=5 μm. Figures represent results from three independent experiments and at least 100 cells were analyzed for each condition. (D) Co-immunoprecipitation (co-IP) of gamma-H2AX with AIM2-Flag in irradiated macrophages using anti-Flag M2 agarose beads. The immunoprecipitates (Flag IP) or the total lysates were analyzed by immunoblotting with antibodies against gamma-H2AX(Ser139) or the Flag tag. Samples from untagged WT mice were used as controls to determine the specificity of immunoblots. IB, immunoblotting. Non-specific band is indicated with an asterisk. Data represent two independent experiments.