Rabbit anti erbb4

Western blot was performed as our previous study (Xu et al., 2019 (link)). In brief, the L3–L5 spinal tissues were collected and homogenized in a RIPA lysis buffer under deep anesthesia with Avertin. Protein concentrations were quantified by the Enhanced BCA Protein Assay Kit (Beyotime Biotechnology), and all samples were adjusted to 4.0 mg/mL. The extracted protein was boiled for 5 min at 95°C with 5 × loading buffer (Beyotime Biotechnology), and an equal volume of the protein mixture was loaded onto an SDS-PAGE gel and transferred onto PVDF membranes in a Western blot system (Bio-Rad, Hercules, CA, United States) at an appropriate voltage and duration. The membranes were blocked with 5% non-fat milk for 1 h at RT before being probed with the primary antibodies: rabbit anti-ErbB4 (1:1000, Cell Signaling Technology, United States), mouse anti- ErbB4 (1: 500, Santa Cruz Biotechnology, United States), or rabbit anti-Neuregulin-1 (1: 500, Santa Cruz Biotechnology, United States) at 4°C overnight followed by incubation with goat anti-rabbit or goat anti-mouse HRP-conjugated secondary antibodies (1:4000; Abbkine) for 1 h at RT. Immunoblots were visualized with a chemiluminescence system (Peiqing Science and Technology, China). Densitometry of the selected bands was determined using ImageJ software (NIH, Bethesda, MD, United States).

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% BSA) 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).

Mouse hippocampal tissue lysates were prepared and subjected to western blot (WB) as previously described [36 (link)]. The primary antibodies used were as follows: mouse-anti β-actin (1:2000, Sigma, A5441), rabbit-anti ErbB4 (1:1000, Cell Signaling Technology, 4795), mouse-anti NRG1 (1:200, Santa Cruz Biotechnology, sc-393006), rabbit-anti GAD65 + 67 (1:2000, Abcam, ab11070), mouse-anti vGAT (1:1000, Synaptic Systems, 131011), rabbit-anti PSD-95 (1:1000, Abcam, ab18258), rabbit-anti Syn-1 (1:1000, Abcam, ab64581). The bands were visualized with the Gel-Pro system (Tanon) and quantified using ImageJ Fiji software.