Iblot dry transfer system

Lysates were collected in 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8.0,
10% glycerol, 0.1% SDS, and 0.5% Na-deoxycholate supplemented with the protease
and phosphatase inhibitor HALT (Thermo Fisher Scientific). Pre-aliquoted SDS and
DTT from New England Biolabs (B7703) were added to the lysate, and proteins were
denatured at 100˚C for five minutes. Proteins were then separated by
SDS-PAGE and transferred to a nitrocellulose membrane via either the iBlot dry
transfer system (Invitrogen) or overnight at 30 V in transfer buffer containing
10% MeOH and 1× Tris-Glycine. Antibody binding was detected using the
LI-COR Odyssey IR imaging system (LI-COR Biosciences).

Whole spinal cord from Ipsilateral T13/L1 was sonicated in a mixture containing extraction buffer (Invitrogen) and protease inhibitors (Sigma). Ice-cold tissue samples were centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was removed, and the protein concentration for each sample was quantified using the Bradford method. Samples were heated to 75°C for 10 min and loaded into a standard polyacrylamide Bis-Tris gel (Invitrogen). SDS-PAGE was performed in 3-(N-morpholino)-propanesulfonic acid running buffer (Invitrogen) at 175 V for 1.25 h. Protein was transferred onto a nitrocellulose membrane using the iblot dry transfer system (Invitrogen). The membrane was blocked with Odyssey blocking buffer (LI-COR Biosciences) for 1 h and incubated with a primary antibody in blocking buffer overnight at 4°C. The following day, the membrane was washed in 1× PBS containing Tween 20 (0.1%) and then incubated in blocking buffer containing either goat anti-rabbit (NLRP3) or goat anti-mouse (B-actin) (LI-COR) IRDye 800CW secondary antibody at a concentration of 1:10,000 (LI-COR) for 1 h at room temperature. Protein expression was quantified using an Odyssey Infrared Imager (LI-COR) and expressed as a ratio to their housekeeping protein. Primary antibodies included rabbit anti-rat NLRP3 monoclonal antibody (1:1000; Abcam), and mouse anti-rat β-actin (1:200,000; Sigma-Aldrich).