Sc 514531

Total protein was extracted using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) from heart tissue and HL-1 cells, and the concentration of the protein was detected by the BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Next, about 20 µg protein was subjected to 12% SDS-PAGE and then transferred on PVDF membrane (Sangon, Shanghai, China). The PVDF membranes were washed with water and stained with Ponceau S to confirm transfer, after that blocked with TBST solution contained 5% skimmed milk at 4 °C overnight. Later, PVDF membranes were incubated with primary antibodies at 4 °C overnight followed by incubated with secondary antibody Goat Anti-Mouse IgG H&L(HRP) (1:10000, ab205719, Abcam, Cambridge, UK) at 25 °C for 2 h. The protein bands were imaged by chemiluminescence apparatus (Shanghai Qinxiang, China) and quantified by Image J software. Primary antibodies including: Wnt1 (1:1000, sc514531, Santa Cruz), β-catenin (1:1000, 8480T, Cell Signaling Technology), IL-10 (1:1000, bs-0698R, Bioss), NOS2 (1:1000, sc7271, Santa Cruz), CD63 (ab68418, Abcam, Cambridge, UK), ACSL4 (1:10000, ab155282, Abcam, Cambridge, UK), TFRC (1:1000, ab214039, Abcam, Cambridge, UK), GAPDH (1:20000, 60004-1-Ig, Proteintech).