Apoptosis-associated speck-like protein containing a CARD pyroptosome were performed following the procedure of (Fernandes-Alnemri and Alnemri, 2008) with minor modifications. Thus, astrocytes were seeded in 50-mm diameter plates (1 × 106 cells per well) and treated with different stimuli. Cells were pelleted by centrifugation and resuspended in 0.5 ml of ice-cold buffer containing PBS/Triton 0.5%, and lysed by shearing 10 times. Cell lysates were then centrifuged at 8000 × g for 15 min at 4°C, and the resultant pellets were washed twice with PBS and resuspended in 200 μl of PBS. The resuspended pellets, were then cross-linked with fresh disuccinimidyl suberate (DSS; 2 mM) for 30 min at room temperature, and pelleted by centrifugation at 8000 × g for 15 min. The cross-linked pellets were resuspended in 30 μl of SDS sample buffer, separated using 12% SDS-PAGE and immunoblotted employing anti-mouse ASC antibodies. The dimer band was quantified by densitometry.
