Pa5 99390

ScN segments of the SNTM bridge (2 mm length) were dissected precisely under a stereomicroscope (Olympus, SZ61) and then subjected to protein extraction. We used direct homogenization and laemmli buffer to isolate and lyse protein samples. We used 25-gauge needles to aspirate protein lysates and subsequently centrifuged the aspiration at 14,000 rpm for 10 min.
We used β-mercaptoethanol, glycerin, and bromophenol-blue to mix and dissolute the collected supernatants and incubated at 95 °C for 5 min. Subsequently, equal amounts of protein samples were resolved in 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride (PVDF) membranes. We blocked these membranes in 5% nonfat dry milk for 2 h at room temperature and probed with primary antibodies: anti-Pro-Caspase-1 (1:1000; Abcam, ab179515), anti-Caspase-1 (1:1000; Invitrogen, PA5-99390), or anti-GSDMD (1:400, Abcam, ab219800), or anti-β actin (1:2000; ab8226, Abcam) at 4 °C overnight. Subsequently, the membranes with primary antibodies were incubated with HRP-conjugated secondary antibodies (1:5000; Pierce, Rockford, IL, United States) for 1 h at room temperature and then developed with enhanced chemiluminescence reagent (Pierce). The quantification of band intensity was performed by using Bio-Rad ChemiDoc XRS + System.

Protein was extracted from the tissue homogenate and macrophages using RIPA lysis buffer (Sigma-Aldrich). NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) were used to prepare the cytosolic fraction and nuclear extracts. Equivalent quantities (25 µg) of protein were separated by SDS-PAGE gel, transferred onto a nitrocellulose membrane (Millipore), blocked with 5% skim milk overnight at 4 °C, followed by overnight incubation with primary antibodies against Atox1 (1:10000; Abcam, ab154179), p47phox (1:1000; Abcam, ab181090), NLRP3 (1:500; Abcam, ab263889), Caspase 1 p20 (1:1000; Invitrogen, PA5-99390), iNOS (1:20000; Abcam, ab178945), IL-12p40 (1:1000; Abcam, ab133752), IL-10 (1:2000; Abcam, ab1333575), Arg-1 (1:1000; Abcam, ab2333548), Lamin B1 (1:1000; Abcam, ab229025), and β-actin (1:5000; Proteintech Group, Inc., 60066-1-AP) at 4 °C. Subsequently, the membranes were washed thrice with Tris-buffered saline with 0.1% Tween-20 and incubated with the HRP-conjugated secondary antibody (1:10000; ZSGB-BIO, ZB-2301, ZB-2305) for 1 h at 37 °C. Signals were visualized with an enhanced chemiluminescence system.