Receptor Purification and Analysis

Dot blots were used to analyze the relative solubility of different samples, while western blots and silver stains were used to detect the receptors and analyze their purity as previously described^{42 (link),43 (link)}. Samples were loaded into Novex 10% Bis–Tris Gels (Life Technologies, Waltham, MA) according to the manufacturer’s instructions, with the exception that the samples were incubated at room temperature for 10 min as boiling can cause receptor aggregation. After electrophoresis, the samples were transferred to a nitrocellulose membrane, blocked with milk (5% w/v non-fat dried milk in TBST) for 1 h, and incubated with the rho1D4 monoclonal antibody (1:3000 in TBST, 1 h at room temperature or overnight at 4 ºC). A goat anti-mouse HRP-conjugated secondary antibody (Pierce, Rockford, IL) and the ECL-Plus Kit (GE Healthcare, Pittsburgh, PA) were used to visualize the receptors. The SilverXpress kit (Life Technologies) was used according to the manufacturer’s instructions to do total protein stains. All images were captured on a Biorad GelDoc system. ImageJ was used to compare sample intensities and to analyze sample purity.