Wnt Signaling Pathway Protein Analysis

Total proteins from H9C2 cells lysed using the radioimmunoprecipitation assay buffer were quantified using a bicinchoninic acid kit (Thermo Scientific, USA). Proteins (20 µL) were loaded and subjected to gel electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Membranes were then exposed to the blocking buffer for 1 h at room temperature and incubated with Wnt3a (ab234099, 1:500; Abcam, UK), Wnt5a (ab174963, 1:500; Abcam), β-catenin (ab32572, 1:500; Abcam), and GAPDH (ab8245, 1:1000; Abcam) antibodies with agitation at 4°C overnight. Subsequently, the membranes were incubated with secondary antibodies (ab6721, 1:2000; Abcam) for 1 h. Enhanced chemiluminescence western blotting substrate (Bio-Rad, USA) was used to detect the signals [25 (link)].