α casp1

The supernatants of the treated cells were precipitated by the chloroform–methanol method, as previously described [34 ]. Pellets were obtained by lysing cells in 25 mM of Tris-HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, and 1% sodium dodecyl sulfate (SDS), plus protease and phosphatase inhibitors. The protein concentration was determined by the BCA protein assay. Samples were separated in a 14% SDS-polyacrylamide gel electrophoresis gel, transferred to a nitrocellulose membrane, and blocked with 5% skimmed milk for 1 h. Then the membrane was incubated overnight with primary antibodies: α-casp1 (1:1000; Cell Signaling 2225), α-IL-1β (1:1000; Acris R1130P), α-NLRP1 (1:1000; ALX-210-904-R100), α-NLRP3 (1:1000; Cell Signaling 15101), α-ASC (1:1000; Santa Cruz Biotechnology sc-22514-R), α-gapdh (1:1000; Cell Signaling 2118), and α-tubulin (1:3000; Abcam ab6160) at 4 °C. Membranes were washed and probed with the appropriate secondary antibody conjugated with peroxidase for enhanced chemiluminescence detection (Amersham Pharmacia Biotech).

The supernatants of the treated cells were precipitated using the chloroform–methanol method, as described by De Nardo. et al. [72 ]. Pellets were obtained by lysing cells in 25 mM of Tris-HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, and 1% SDS, plus protease and phosphatase inhibitors. BCA protein assay was used to determine protein concentration. Samples were separated in a 14% SDS-PAGE gel, transferred to a nitrocellulose membrane, and blocked with 5% skimmed milk for 1 h. Then, the membrane was incubated overnight with primary antibodies: α-casp1 (1:1000; Cell Signaling 2225), α-IL-1β (1:1000; Cell Signaling #82186S and Merck Millipore #MAB18), α-NLRP3 (1:1000; Cell Signaling 15101), α-ASC (1:1000; sc-22514), α-GSDMD (1:1000; Cell Signaling #93709), and α-GAPDH (1:1000; Cell Signaling 2118S) at 4 °C. Membranes were washed and probed with the appropriate secondary antibody conjugated with peroxidase for enhanced chemiluminescence detection (GE Healthcare Bio Sciences AB, Uppsala, Sweden).