Nbp2 12446

After deparaffinization and dehydration, tissue sections were incubated overnight at 4°C with NLRP3(Novus Biologicals, NBP2-12446), DLST(Abcam, ab177934), SLC31A1 antibodies (Proteintech, 67221-1-Ig) after epitope retrieval, H₂O₂ treatment, and non-specific antigens blocking. Then the sections were treated for two hours at room temperature with secondary antibodies (Proteintech, SA00001-2), and an enhanced DAB staining kit (Vector, SK-4100) was used to detect the signal. The ImageJ software was used for histomorphometric evaluation.

Whole-cell lysates were obtained and protein concentrations determined by a BCA protein assay (Thermo Fisher Scientific, Rockfold, IL). Protein was separated by SDS-PAGE gel electrophoresis and subsequently transferred to a PVDF membrane (EMD Millipore) using a Bio-Rad semi-dry transfer cell (20 V, 1 h). Blots were incubated with anti-GPR43 (Millipore, ABC299, 1.0 μg/ml), NLRP3 (Novus Biologicals, NBP2-12446, 5.0 μg/ml), Caspase-1 (Abcam, ab179515, 1:1000 dilution), IL-18 (MBL, D046-3, 1.0 μg/ml), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204, Cell Signaling Technology, 9101, 1:1000 dilution), p44/42 MAPK (Erk1/2) (137F5, Cell Signaling Technology, 4695, 1:2000 dilution) or β-actin (Abcam, ab8226, 1:3000 dilution) primary antibodies overnight at 4 °C. Incubation with secondary anti-rabbit-HRP (Santa Cruz, sc-2357), anti-mouse-HRP (Santa Cruz, sc-2005), or anti-rat-HRP (Abcam, ab97057) was performed at room temperature for 1 h. Bound antibody was detected using SuperSignal ECL substrate (Thermo Fisher Scientific). Densitometric analysis was performed using ImageJ.

After deparaffinization and dehydration, tissue sections were incubated overnight at 4°C with NLRP3(Novus Biologicals, NBP2-12446), DLST(Abcam, ab177934), SLC31A1 antibodies (Proteintech, 67221-1-Ig) after epitope retrieval, H₂O₂ treatment, and non-specific antigens blocking. Then the sections were treated for two hours at room temperature with secondary antibodies (Proteintech, SA00001-2), and an enhanced DAB staining kit (Vector, SK-4100) was used to detect the signal. The ImageJ software was used for histomorphometric evaluation.

Total protein was extracted from left atrial tissue and atrium fibroblasts using RIPA Lysis Buffer (Beyotime, P0013B, Beijing, China). Protein concentration was measured with the BCA Protein Assay kit (Beyotime, P0010, Beijing, China). Protein (25 μg) was separated via 10% SDS-PAGE at 70 V for 1 h and then transferred onto PVDF membranes at 220 mA for 2 h. The membranes were blocked with 5% nonfat powdered milk in tris-buffered saline with tween (TBST) for 1 h at room temperature. The blot was incubated overnight at 4°C with primary antibodies targeting TLR4 (Abcam, ab22048, diluted 1 : 1000), NLRP3 (NOVUS, NBP2-12446, diluted 1 : 250), pro-caspase-1 (Abcam, ab179515, diluted 1 : 1000), caspase-1-p20 (Proteintech, 22915-1-AP, diluted 1 : 1000), TGF-β (Cell Signaling Technology, 3711, diluted 1 : 1000), IL-1β (Cell Signaling Technology, 31202, diluted 1 : 1000), IL-18 (Wanleibio, diluted 1 : 1000, Shenyang, China), collagen I (Proteintech, 14695-1-AP, diluted 1 : 2000), or GAPDH (Cell Signaling Technology, 5174, 1 : 2000). The membranes were then washed with TBST three times and incubated with the corresponding secondary antibodies for 1 h at room temperature. GAPDH was used as an internal control. The western blot bands were imaged by an enzymatic chemiluminescence (ECL) kit (Solarbio, PE0010, Beijing, China) and quantified using ImageJ software.