Ab239510

Total protein was extracted by Whole-Cell Lysis Assay (KeyGEN BioTECH) from NRCMs and heart tissues, and the concentration was defined by the BCA Protein Assay Kit (KeyGEN BioTECH). Protein extractions were boiled and denatured, separated by 10% SDS–PAGE and then transferred onto PVDF membranes (Millipore). After blocking with 5% skimmed milk powder, the PVDF membranes were incubated overnight at 4 °C with the following rabbit-sourced primary antibodies, including anti-METTL3 antibody (Abcam, ab195352, 1:1000), anti-ANP antibody (Abcam, ab189921, 1:1000), anti-BNP antibody (Abcam, ab239510, 1:1000), anti-DKK2 antibody (Abcam, ab95274, 1:1000), anti-β-catenin antibody (Abcam, ab32572, 1:1000), anti-c-Myc antibody (Abcam, ab32072, 1:1000), and anti-β-actin antibody (ABways, AB0035, 1:5000). Subsequently, the membranes were washed with TBST and incubated with HRP-conjugated secondary antibody (Abways, AB0101, 1:5000; AB0102, 1:2000) at room temperature for 1 h. Finally, bands were detected using ECL (KeyGEN BioTECH) with a chemiluminescence system (Bio-Rad).

Hearts were fixed in 4% paraformaldehyde, embedded in paraffin, and dissected into 5-μm-thick serial transverse sections at the papillary muscle levels. Hematoxylin and eosin staining, and Masson’s trichrome staining were used to evaluate heart tissue morphology. Sirius red staining was performed to visually assess cardiac fibrosis extents. Immunohistochemical staining was conducted using anti-BNP antibody (1:500) (Abcam, ab239510) for 24 h at 4 °C and horseradish peroxidase-linked secondary antibody (1:500) (Abcam, ab97023) for 60 min at room temperature. And further immunohistochemistry steps were conducted as previously reported methods. Images were captured by a microscope (BX61TRF, Olympus, Japan) and quantified using Image J software (Ver 1.51, National Institutes of Health) and Image-Pro Plus 6.0 software (Media Cybernetics Inc.).