Bsm 33207m

Total protein samples were isolated from tissue samples using RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) containing both PMSF and phosphatase inhibitors for western blot analyses. A BCA Protein Assay Kit (23225, Thermo Fisher Scientific, Rockford, IL, United States) was used to determine the protein concentration. The protein samples were separated on the 10% or 12% SDS-PAGE gels, and then transferred to a polyvinylidene fluoride (PVDF, Millipore, MA, United States) membranes. After blocking with 5% skim milk, the PVDF membranes were incubated overnight at 4°C with the following primary antibodies: anti-TNFR1 (1:1000, 21574-1-AP, Proteintech, China), anti-phospho-NF-κB p65(Ser536) (1:1000, 8324, CST), anti-IL1β (1:1000, ab6722, Abcam, United States), IL6 (1:1000, bs-6309R, Bioss), iNOS (1:1000, bs-0162R, Bioss), anti-TNFα (1:1000, bsm-33207M, Bioss), anti-SREBP-1 (1:1000, sc-365513, Santa Cruz Biotechnology), anti-PPARγ (1:1000, bsm-4590R, Bioss) and β-actin (1:5000, AC026, ABclonal, China). After that, the PVDF membranes were incubated with the corresponding secondary antibodies. An ECL kit (Tanon, Shanghai, China) was used to visualize the chemiluminescence signals. Protein expression levels were quantified with ImageJ software. The housekeeping protein β-actin was utilized as loading control.

Five rats were randomly selected from each group (10 rats in total), and the myocardium at the apex of the heart was obtained for western blot detection. This experiment was performed to observe the relative expression of the following proteins in myocardial tissue: TNF-α (bsm-33207 m; Bioss), sprouty RTK signaling antagonist 2 (SPRY2; GB11860; Servicebio), receptor for advanced glycosylation end-product specific (RAGE; 16,346–1-AP; Proteintech), lamin B1 (GB1111802; Servicebio), lamin A/C (GB11407, Servicebio), IL-6 (GB11117, Servicebio) and CACNA1C (21774-1-ap; proteintech). The experimental method of this part is in the Additional file 3: Experimental methods.