Nlrp3

Samples were obtained from the border zone either at day 3 in Experiment 1 or 1 hour in Experiment 2 after MI. Antibodies to p65 NF‐κB (Santa Cruz Biotechnology), NLRP3 (Santa‐Cruz Biotechnology, sc‐34408), cleaved IL‐1β (Cell Signaling Technology) and β‐actin (Santa Cruz Biotechnology) were used. Western blotting procedures were described previously.¹¹ Experiments were replicated three times and results expressed as the mean value.

Lung tissues were homogenized, separated on 12% SDS-PAGE gel, and then transferred onto a PVDF membrane. The membrane was blocked in 5% fat-free milk and probed with primary antibody against pro-IL-1β (Cell Signaling Technology, USA), IL-1β p17, NLRP3, caspase-1 p10 (Santa Cruz, USA) and IκB (Beyotime Biotechnology, China). Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, USA; Cell Signaling Technology, USA) and enhanced chemiluminescence were applied to detect protein content. Images were collected using ChemiDoc XRS (Bio-Rad, USA). Bands were quantified using Image J.

Protein expression was evaluated by western blotting, as described previously [23 (link)]. Antibodies specific for NF-κB p65 (#8242; Cell Signaling Technology, Danvers, MA, USA), NLRP3 (#sc-66846; Santa Cruz Biotechnology, Santa Cruz, CA, USA), pro-caspase-1 (#IMG-5028; Novus Biologicals, Littleton, CO, USA), caspase-1 (#sc-56036; Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (#ab9722; Abcam, Cambridge, MA, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#2118; Cell Signaling Technology, Danvers, MA, USA), ASC (#ab180799; Abcam, Cambridge, MA, USA), gasdermin D (GSDMD) (#ab210070; Abcam, Cambridge, MA, USA), HBx (#ab39716; Abcam, Cambridge, MA, USA), hepatitis B virus core protein (HBc) (#MAB16989; Merck Millipore, Billerica, MA, USA), and cytochrome c oxidase subunit 4 (COXIV) (#GB11250; Wuhan Goodbio Technology, Wuhan, China) were used.

The cells were lysed in buffer containing 10 mM Tris-buffer (pH 7.6), 1% Triton X-100, 1% phosphatase inhibitor cocktail and 1 mM PMSF. The lysates were boiled in sodium dodecyl sulfate (SDS) sample buffer and were subjected to SDS–PAGE. Cytoplasmic extracts and nuclear extracts were obtained using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Sigma, 78833). Rabbit monoclonal antibodies against GAPDH, NLRP3, and ASC were purchased from Santa Cruz Biotechnology (CA, USA) and were diluted 1:1000. The anti-ATG5 antibody, anti-LC-3 antibody, anti-Caspase-1 antibody, anti-PCNA, and anti-p65 antibody were purchased from Cell Signaling Technology (Beverly, MA, USA) and were diluted 1:1000. The anti-TRIF antibody was purchased from Abcam and was diluted 1:1000. Horseradish peroxidase- conjugated goat anti-rabbit immunoglobulin G (Cell Signaling Technology, MA, USA) was used as the secondary antibody. Immunoreactive bands were identified using the ECL Western Blotting Detection System (Millipore Corporation, Billerica, MA, USA).

Proteins extracted from kidney or cultured HK-2 cells were separated by 10–12% SDS-PAGE, transferred onto PVDF membrane, and blocked with 5% nonfat milk. The PVDF membranes were incubated with 1^st antibodies of KIM (1:500, Abcam, USA), collagen I (1:1000, Santa Cruz Biotechnology), collagen IV (1:1000, Santa Cruz Biotechnology), FN (1:1000, Santa Cruz Biotechnology), α-SMA (1:1000, Santa Cruz Biotechnology), E-cadherin (1:1000, Cell Signaling Technology), NLRP3 (1:1000, Santa Cruz Biotechnology), ASC (1:1000, Santa Cruz Biotechnology), Caspase-1 (1:1000, Cell Signaling Technology and IL-1β (1:1000, Cell Signaling Technology) overnight at 4 °C. After washing three times with TBST, the PVDF membranes were incubated with 2^nd antibodies at room temperature for 1 h, and then the protein bands were observed by enhanced chemiluminescence (ECL) (Thermo, Rockford, USA). Densitometric analysis was performed using the Bio-Rad Quantity One Software. The relative expression of each target protein was normalized to the GAPDH band.

The total DNA/RNA/Protein Kit (R6734, Omega Bio-tek, Norcross, GA, USA) was utilized to extract total protein from hippocampal tissue according to the manufacturer’s instructions. Western blot was conducted for CREB (#9197, Cell signaling technology, USA), p-CREB (#9198, Cell signaling technology, USA), BDNF (ab108319, Abcam, UK), AKT (#9272, Cell signaling technology, USA), p-AKT (#4060, Cell signaling technology, USA), PSD95 (#3450, Cell signaling technology, USA), GABAA (GB113973, Servicebio, Wuhan, China), Synapsin-1 (SYN, #5297, Cell signaling technology, USA), Bax (#2772, Cell signaling technology, USA), Bcl-2 (#3498, Cell signaling technology, USA), Caspase-3 (#9662, Cell signaling technology, USA), Cleaved-caspase 3 (#9661, Cell signaling technology), NF-kB (P65) (#8242, Cell signaling technology, USA), NLRP3 (sc-134306, Santa Cruz, CA, USA), Caspase 9 (#9504, Cell signaling technology), Caspase 1 (#3866, Cell signaling technology), β-actin (bs-10966R, Bioss, Beijing, China) of hippocampal total protein. The density of protein bands was assessed using a computing densitometer. IL-1β was measured using ELISA kits (Nanjingjiancheng, Nanjing, China) according to the manufacturer’s protocols.