Anti phosphoerbb3

For in vitro NRG1 processing experiments 293 cells were transfected with NRG1 type I or III and treated with 20 ug/ml antibodies for 72 hours. For evaluation of signaling in brains of anti-NRG1-treated mice, wild type or ErbB4^−/− mice were treated with anti-NRG1 or anti-ragweed (20 mg/kg, i.p., 2 dose). Treated cells from in vitro experiments of hippocampus of antibody treated mice were lysed in cold RIPA buffer containing protease and phosphatase inhibitor cocktails (Thermo Scientific) using homogenizer. Protein concentration was determined by BCA (Thermo Scientific). Proteins were separated by SDS-PAGE and transferred to nitrocellulose by iBlot. Primary antibodies used were anti-actin (BD Biosciences), anti-NRG1, anti-ErbB3 and anti-ErbB4 (Santa Cruz Biotechnology), anti-phospho-ErbB3, anti-phospho-ErbB4 (Cell Signaling Technology), anti-Cofilin and anti-phospho-Cofilin (Novus Biological). The therapeutic antibody was not capable of recognizing denature antigen and therefore, could not be used for immunoblot analysis. Secondary antibodies were IRDye 680 conjugated goat anti-mouse IgG and IRDye 800 CW conjugated goat anti-rabbit IgG (Li-Cor Biosciences). Images were recorded and band intensities determined by LICOR.