Nlrp3

Our recent study has described this method detailly [2 (link)]. The primary antibodies used in this present study included NOX2 (ab129068, 1 : 2000, Abcam, USA), AT1 (381666, 1 : 1000, Zenbio, Chengdu, China), ACE (24743-1-AP, 1 : 1000, proteintech, China), Bax (200958, 1 : 1000, Zenbio, Chengdu, China), MMP13 ((820098, 1 : 1000, Zenbio, Chengdu, China), C-caspase-3 (#9664, 1 : 1000, Cell Signaling Technology, Inc. USA), p53 (sc-393031, 1 : 500, Santa Cruz, Bio. Lnc, USA), Klotho (382164, 1 : 1000, Zenbio, Chengdu, China), Bcl2 (381702,, 1 : 1000, Zenbio, Chengdu, China), COX-2 (#12282, 1 : 1000, Cell Signaling Technology, Inc. USA), MMP-3 (380816, 1 : 1000, Zenbio, Chengdu, China), aggrecan (ab36861, 1 μg/mL, Abcam, USA), iNOS (#20609, 1 : 1000, Cell Signaling Technology, Inc. USA), Nrf2 (221102, 1 : 1000, Zenbio, Chengdu, China), HO-1 (#43966, 1 : 1000, Cell Signaling Technology, Inc. USA), type II collagen (collagen II (1 : 1000, ab34712, Abcam), SOD1 (#37385, 1 : 1000, Cell Signaling Technology, Inc. USA), NLRP3 (381207, 1 : 1000, Zenbio, Chengdu, China), ASC (340097, 1 : 1000, Zenbio, Chengdu, China), and GAPDH (5174, 1 : 1000, Cell Signaling Technology, Inc. USA). The secondary antibodies were purchased from Zenbio (380172, 511103, Zenbio, Chengdu, China).

The protein concentration in the hippocampus or cells was detected using a BCA kit (Biyuntian Institute of Biotechnology, China) and adjusted to the same level. The samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene PVDF membrane (Millipore, Bedford, MA, United States). After being blocked with 5% skim milk for 2 h, the membrane was incubated with primary antibodies overnight at 4°C and secondary antibodies for 2 h at room temperature. The specific primary antibodies were against SIRT1 (1:1,000, Proteintech Group, INC., China), FOXO3A (1:1,000, Proteintech Group, INC., China), and NLRP3 (1:1,000, Zen-Bioscience, China). The secondary antibodies were the horseradish enzyme-labeled goat antimouse secondary antibody (1:1,000, ZhongShan Jinqiao Biotechnology Co., Ltd., China) and the horseradish enzyme-labeled goat antirabbit secondary antibody (1:1,000, ZhongShan Jinqiao Biotechnology Co., Ltd., China). The protein bands on the membrane were recorded using a gel imaging system (UVP, California, United States).

First, colon tissues were homogenized with glass beads (60 Hz, 30 + 30 s), then lysed using the RIPA&PMSF lysis buffer (Beijing solarbio technology & generation Co., Ltd, China). Lysates were centrifuged at 12,000 rpm for 5 min. The supernatant was collected and mixed with a five-fold sodium dodecyl sulfate (SDS) buffer. Samples were separated by 6–12% acrylamide gel electrophoresis and transferred to the polyvinylidene fluoride film. After incubation with primary and secondary antibodies, protein bands were detected by VILBER Fusion FX7 and analyzed by ImageJ.
The following antibodies were used: ZO-1 Rabbit polyclonal antibody (Cat.No. YN1410, Immunoway), Occludin Rabbit polyclonal antibody (Cat.No. 381549, ZENBIO), NLRP3 Rabbit polyclonal antibody (Cat.No. 381207, ZENBIO), caspase-1 Rabbit polyclonal antibody (Cat.No. 342947, ZENBIO), ASC Rabbit polyclonal antibody (Cat.No. 340097, ZENBIO), IL-1β Rabbit polyclonal antibody (Cat.No. TA5103, Abmart), β-actin Rabbit polyclonal antibody (Cat.No. AC006, Abclonal).