Anti baff

The B-cells were lysed in protein lysis buffer containing a proteinase inhibitor (Thermo Fisher Scientific). Lysates were then centrifuged for 15 min at 14,000g at 4°C, and the protein concentration was determined using the Bradford Protein Assay (Thermo Fisher Scientific). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 10% polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 buffer and immunoblotted with primary antibodies, including anti-KLF5 (Abcam, Cambridge, UK), anti-BAFF (Abcam), and anti-GAPDH (Cell Signaling, BSN, USA). The band intensity was then quantified using Quantity One software (Bio-Rad, CA, USA).

The ventricle tissues were homogenized, and total proteins obtained using Tissue and Cell Total Protein Extraction Kits (Jiangsu KeyGEN BioTECH Corp., Ltd) were used to analyse BAFF and TNF‐α expression. Total protein (20 μg) was resolved via 15% SDS‐PAGE (Beyotime Biotech Co., Ltd., Shanghai, China) and then transferred to polyvinylidene fluoride membranes. Nonspecific protein binding on the membranes was blocked with 5% non‐fat milk for 2 h, followed by incubation with anti‐BAFF (Abcam plc.), anti‐TNF‐α (Proteintech Group, Inc.) and anti‐β‐actin (Cell Signalling Technology Inc.) antibodies overnight at 4°C. The anti‐β‐actin was selected as reference according previous study¹⁸ and due to the molecular weight of BAFF being close to that of anti‐GAPDH. After three washes with TBST buffer, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibody at room temperature for 2 h. Proteins were detected using an enhanced chemiluminescence system and quantified using image J software.

The above THP-1 cells were lysed in protein lysis buffer containing a proteinase inhibitor (Thermo Fisher Scientific). Lysates were then centrifuged for 15 min at 14,000g at 4°C, and the protein concentration was determined using the Bradford Protein Assay (Thermo Fisher Scientific). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 10% polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 buffer and immunoblotted with primary antibodies, including anti-BAFF (Abcam) and anti-β-actin (Cell Signaling, BSN, USA). The band intensity was then quantified using Quantity One software (Bio-Rad, CA, USA).