Ag1478

The recombinant human Neuregulin-1/Heregulin-β1 (NRG1, ProSpec) polypeptide chain (a.a 177-241) and ecto-ErbB4, a neutralizing peptide blocking NRG1 activation of ErbB kinases, were prepared in 0.1% BSA (vehicle). AG-1478 (ApexBio), an ErbB4 inhibitor, was dissolved in DMSO (Sigma-Aldrich). The drug doses were selected on the basis of previous reports and our preliminary studies. NRG1 (Lacroix-Fralish et al., 2008 (link); Calvo et al., 2010 (link)), ecto-ErbB4 (Woo et al., 2007 (link)) and AG-1478 (Araldi et al., 2018 (link)) were delivered intrathecally at concentration of 10 nM, 1 μg/mL, and 5 μM in a volume of 2 μL, respectively, for 3 consecutive days after intraplantar injection of CFA. Tamoxifen was administrated following our previous protocols with modification (Wang et al., 2018a (link)). Simply, Tamoxifen (Sigma-Aldrich) was dissolved in corn oil at 20 mg/ml. Mice (8 weeks old) were intraperitoneally injected with Tamoxifen at 100 mg/kg/day (i.p.) for 5 consecutive days.

Tg(fabp10a:pt-β-catenin) larvae were treated with 0.1% dimethyl sulfoxide (DMSO), 1 µM GW7647 (Cayman Chemical, Ann Arbor, MI), 2 µM K-975 (MedChemExpress, Princeton, NJ), 15 µM Etomoxir (Cayman Chemical, Ann Arbor, MI), 2 µM AG1478 (ApexBio, Houston, TX), or 7 μM CAY10599 (Cayman Chemical, Ann Arbor, MI) for 48 h from 12 or 13 days post-fertilization (dpf). Compounds were refreshed every 24 h. The larvae were fed prior to the drug treatments but fasted during the treatments. They were harvested at 14 or 15 dpf for subsequent analyses.

AG1478, Erlotinib, and SB225002 were purchased from ApexBio, Biovision, and Enzo Life Science, respectively. Recombinant human IL-1β, CXCL1, and IL-1Ra was purchased from R&D System. Western blotting analysis was performed using antibodies against human EGFR and phosphorylated EGFR (pEGFR) (Y1068) (Cell Signaling). Neutralizing antibodies experiments were performed using antibodies against human IL-1β (Acris Antibodies) and human CXCL1 (RayBiotech).