The protein extracts from the cortex and cultured astrocytes were homogenized in 250 mg tissue/0.5 ml cold lysis buffer (see above). Then they were kept on ice for 30 min, centrifuged at maximum speed for 15 min, and the supernatant was collected to determine the proteins levels using the Bradford Assay (Bio-Rad, Hercules, CA, USA). Lysates were separated by SDS-PAGE gels and were transferred to PVDF membranes following standard techniques. Membranes were blocked with 5% non-fat dried milk in TBS containing 0.1% Tween-20 (TBS-T). Next they were then incubated overnight with the following primary antibodies: anti-NLRP3 (1 μg/ml, Abcam), anti-caspase-1 (1/100, Santa Cruz Biotechnology); anti-pro-caspase-1 (1/200, Abcam); anti-Apaf-1 (1 μg/ml, Millipore Bioscience Research Reagents); anti-caspase-3 (1:500) and anti-caspase-9 (1:1000, Cell Signaling).
