Mouse anti nlrp3 ag 20b 0014 c100

Brain and gut tissues were collected, flash-frozen, and stored at −80 °C. Methods for SDS-PAGE electrophoresis and Western blotting were adapted from our previous work [43 (link)]. The following primary antibodies were selected: mouse anti-NLRP3 (#AG-20B-0014-C100; Adipogen, San Diego, CA, USA), rabbit anti-Caspase-1 (#A0964; Abclonal, Woburn, MA, USA), or mouse anti-GAPDH (#AM4300; ThermoFisher Scientific, Waltham, MA, USA). Donkey anti-mouse or goat anti-rabbit HRP-labeled IgGs (Vector Laboratories, Burlingame, CA, USA) were used as secondary antibodies. Bands were detected by enhanced chemiluminescence using the SuperSignal™ West Femto substrate (Thermo-Fisher Scientific, Waltham, MA, USA). Membranes were imaged with the Odyssey Fc imaging system (Li-Cor, Lincoln, NE, USA). Signal density was quantified using the complementary software, Image Studio Lite (Li-Cor, Lincoln NE, USA), with the relative expression of NLRP3 and Caspase-1, normalized to endogenous GAPDH levels.
Two-way analysis of variance (ANOVA) was used to determine the effects of genotype and age on biochemical and morphological measures using GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA). Values of p < 0.05 were considered statistically significant, and data are expressed as mean ± SEM.

Western blotting was performed as previously described^{59 (link)}. Briefly, proteins were separated by 6–15% SDS-PAGE and electro-transferred to PVDF membranes. The membranes were blocked in TBST with 5% BSA for 30 min at room temperature, incubated with primary antibodies overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibodies (Bio-Rad) for 1 h at room temperature. After washing, blots were developed using the SuperSignal™ West Dura Extended Duration Substrate (Thermo Scientific) and imaged on a Bio-Rad ChemiDoc Imaging system. Mouse anti-α-tubulin antibody (T5168) and mouse anti-β-actin antibody (A1978) were from Sigma-Aldrich. Rabbit anti-ubiquitin (#3936), anti-cleaved-IL-1β (#63124), anti-NLRP3 (#15101), anti-ASC (#67824), anti-Gasdermin D (#93709) and anti-p62 (#39749) antibodies were from Cell Signaling Technology. Mouse anti-NLRP3 (AG-20B-0014-C100) and anti-Caspase 1 (p20) (AG-20B-0042-C100) antibodies were from Adipogen. Goat anti-IL-1β antibody (AF-401-NA) was from R&D Systems. Rabbit anti-MafB (20189-1-AP) and mouse anti-GAPDH (60004-1-Ig) were from Proteintech. All antibodies were used at a dilution of 1:2000. Protein densitometric analyses were performed using ImageJ using α-tubulin or β-actin as reference.