Western Blot Analysis of Noradrenergic Nuclei

Noradrenergic nuclei from the brainstem were homogenized in lysis buffer (Sigma Aldrich, St. Louis, MO) and protein concentrations were determined using BCA assay as previously described^{12 (link), 13 (link)}. 20μg of protein was loaded for each sample into SDS-PAGE gels (NuPAGE, Invitrogen, Carlsbad, CA). Proteins were then transferred onto PVDF membranes which were probed with antibodies including pSTAT-3 (1:1000; goat-polyclonal; Santa Cruz Biotechnology, Dallas, TX), SOCS-3 (1:1000; goat-polyclonal; Cell Signaling Technology, Danvers, MA), and GAPDH antibody (1:2000; mouse-monoclonal; Sigma-Aldrich, St. Louis, MO). After washing 3 times of 10 min each, the membranes were incubated in blocking solution containing goat anti-rabbit DyLight 800 and goat anti-mouse DyLight 680 secondary antibodies (1:5000; Thermo Fisher Scientific; Waltham, MA). Bands were visualized and analyzed using an Odyssey imaging system (Li-COR biosciences, Lincoln, NE).