Ab95274

A protein extraction kit (Beyotime, Shanghai, China) was used to extract total protein. Then protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). 5% nonfat milk was used to block the membrane, and then the membrane was incubated with the primary antibodies against β-catenin (1:5000; ab32572, Abcam, Cambridge, UK), Ki67 (1:5000; ab92742, Abcam, Cambridge, UK), DKK2 (1:1000; ab95274, Abcam, Cambridge, UK), FRZB (1:1000; ab205284, Abcam, Cambridge, UK) or GAPDH (1:5000; ab181602, Abcam, Cambridge, UK) overnight at 4 °C. Subsequently, the membrane was incubated with the secondary antibody (1:5000; ab205718, Abcam, Cambridge, UK) at room temperature for 1.5 h. The protein signals were showed by enhanced chemiluminescence reagents (Millipore, Billerica, MA, USA).

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).