Western Blot Analysis of Frizzled-1 Signaling

Total ventricular extracts were prepared from the LV myocardium as described previously [31 (link)]. Protein concentrations were measured by BCA (Sigma-Aldrich, USA) assay. Equal amount of protein extracts was separated with SDS-page and transferred to a nitrocellulose membrane (Millipore, USA). The following primary antibodies were used in the present study: anti-GAPDH (USBiological, USA), anti-FZD1 (R&D systems, USA), anti-active-β-catenin (Millipore, USA), anti-β-catenin (Santa Cruz Biotechnology, USA), anti-p-GSK-3β (Cell-Signaling Technology, USA), anti-GSK-3β (BD Biosciences, Germany). HRP-conjugated anti-rabbit IgG, anti-mouse IgG, and anti-goat IgG antibodies (Santa Cruz Biotechnology, USA) were used as secondary antibodies. The blots were analyzed and quantified by densitometry using Image J program.