Caspase 1 antibody

The frozen heart tissues were homogenized and then lysed using RIPA lysis buffer (Beyotime Biotechnology, China) added with the protease inhibitor (Thermo Fisher Scientific, MA). Protein samples were separated by SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with primary antibodies overnight and then with secondary antibodies for another hour. Finally, the bands were visualized using ECL solution (Merk Millipore, MA). The following antibodies were used: CatB antibody (1:200 dilution) from Santa Cruz, NLRP3 antibody (1:1000 dilution) from Abcam, ASC antibody (1:1000 dilution) from Millipore, caspase-1 antibody (1:1000 dilution) from Abcam, β-actin antibody (1:3000 dilution) from Santa Cruz, Horseradish peroxidase (HRP)- conjugated anti-rabbit and anti-mouse IgG (1:3000) from Santa Cruz. Densitometric quantification of the bands were performed using ImagePro Plus (Media Cybernetics, Warrendale, PA).

RHu PDGF-BB, IL-1β and imatinib mesylate were purchased from MedChemExpress, United States (cat. nos. HY-P7055; HY-P7097; HY-50946); PDGF-BB polyclonal antibody, collagen II monoclonal antibody and aggrecan neo polyclonal antibody were purchased from Thermo Fisher Scientific, United Kingdom (cat. nos. PA5-88272; MA5-12789; PA1-1746); MMP3 antibody, MMP9 antibody, Adamts4 antibody, Adamts5 antibody and Caspase1 antibody were purchased from Abcam, United Kingdom (cat. nos. ab52915; ab76003; ab185722; ab41037; ab179515); PDGFR antibody, P-PDGFR antibody, P-P38 antibody and P-JNK antibody were purchased from Hangzhou Huaan Biotechnology Co., Ltd., China (cat. nos. SN0646; R1510-44; ER2001-52; ET1601-28); P-PI3K antibody, PI3K antibody, P-AKT antibody, AKT antibody, P38 antibody, JNK antibody, P-ERK antibody, ERK antibody, β-actin antibody and NLRP3 antibody were purchased from Cell Signaling Technology, United States (cat. nos.17366; 4292; 9271; 4691; 9212S; 9252S; 4370; 4695; 4970; ab263899); IL-1β antibody was purchased from ABclonal Technology Co., Ltd., China (cat. no. A1112).

Total protein was extracted from GMCs using a protein extraction kit (Roche, USA). Protein concentrations were assessed by using BCA Protein Assay Kit (Bioworld Technology, USA). The OD value was measured at a wavelength of 562 nm, the standard curve (r > 0.99) calculated protein sample concentration. Western blotting was performed as previously described [27 (link)]. Immunoblotting was performed using NLRP3 antibody (rabbit, 1 : 800, CST, USA), caspase-1 antibody (rabbit, 1 : 1000, Abcam, USA), procaspase-1 antibody (rabbit, 1 : 1000, Abcam, USA), IL-1β (rabbit, 1 : 800, Abcam, USA), pro-IL-1β (rabbit, 1 : 1500, Abcam, USA), and GAPDH (rabbit, 1 : 10000, Bioworld Technology, USA). The second antibody of NLRP3, caspase-1, procaspase-1, IL-1β, and pro-IL-1β (1 : 2000, anti-rabbit) was obtained from the Beyotime Institute of Biotechnology, Shanghai, China. The proteins were detected with HRP chemiluminescence reagent (Millipore, USA) and images were captured with the UVP imaging system (Bio-Rad, USA).