Quantitative Western Blot Analysis of sFRP4

Protein homogenates were prepared with a standard radioimmunoprecipitation assay buffer supplemented by protease and phosphatase inhibitor cocktails as previously described (17 (link)). Unless otherwise specified, 45 mg of protein per well was loaded onto 12% Bis-Tris polyacrylamide gels (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes. Nonspecific binding was blocked with 5% nonfat dried milk suspended in Tris-buffered saline (pH 7.2) supplemented with 0.1% Tween-20 (Sigma, St. Louis, MO). Polyclonal antibody specific for sFRP4 was obtained from Abcam (Cambridge, MA) and used at 1:1000 dilution. Horseradish peroxidase–conjugated secondary antibody (Sigma-Aldrich, St. Louis, MO) was used at 1:500 dilution. Immunoreactivity was visualized with Amersham ECL Plus Detection Reagents (GE Life Sciences, Piscataway, NJ). GAPDH was used as a loading control for all experiments. Anti-GAPDH polyclonal antibody was obtained from Sigma and used at 1:1000 dilution. Results of Western blots were quantified via a LICOR Odyssey Fc Imaging system, normalized to expression of the GAPDH loading control by defining a uniform region of interest.