Anti erbb4

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.

Anterior right GP was excised and washed with saline at the end of the experiment. Tissue for western blot analysis was dissected into small portions and maintained at -80°C for subsequent analyses. Tissue for histological studies was fixed with 4% paraformaldehyde. The protein expression levels of c-fos and nerve growth factor (NGF) in ARGP were analyzed using western blot analysis. The primary antibodies used were anti-c-fos (Abcam, Cambridge, England) and anti-NGF (Santa Cruz, Dallas, TX, United States). The membrane was washed and incubated with secondary anti-rabbit antibodies at 37°C for 2 h. Antibody-binding protein bands were visualized and quantified. Immunofluorescence staining was used to confirm the expression and location of ErbB4 in atrial GP. Paraffin-embedded GP tissues were cut transversely into 5-μm sections. The primary antibodies used were anti-ErbB4 (Santa Cruz, Dallas, TX, United States) and anti-parvalbumin (Abcam, Cambridge, England). Quantitative analyses were performed using commercially available software (Image Pro Plus, Media Cybernetics, Inc., Rockville, MD, United States).