Mouse anti nrg1

Brain tissues were homogenized in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA) containing 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 1 mM DTT and protease inhibitor cocktail (ThermoFisher, 11697498001). Samples were resolved on SDS-PAGE and transferred to the nitrocellulose membranes (Cat# 1620112, Bio-Rad). After blocking with 5% BSA (bovine serum albumin in PBS-T, PBS containing 0.5% Tween-20), membranes were incubated in primary antibody (in 5% bovine) overnight at 4 °C. Membranes were washed with PBS-T (10 min◊3) and incubated in HRP-conjugated secondary antibody (Life Technology) at RT for 1 h. After wash, the immunoreactive bands were visualized by applying enhanced chemiluminescence substrates (Cat# 32106, Pierce), signals were captured with LI-COR Odyssey infrared imaging system. Antibodies used included: rabbit anti-ErbB4 (1:2000, 0618, generously provided by Cary Lai); rabbit anti-phospho-ErbB4 (1:200, Cat# 4757, Cell Signaling Technology, RRID:AB_2099987); mouse anti-β-actin (1:5000, Cat# A1978, Sigma, RRID:AB_476692); mouse anti-phospho-ERK (1:500, Cat# 9106, Cell Signaling Technology, RRID:AB_331768); rabbit anti-ERK (1:500, Cat# 9102, Cell Signaling Technology, RRID:AB_330744); mouse anti-NRG1 (1:200, Cat# MA5–12896, Invitrogen, RRID:AB_10986946).