Neuroblastoma Cell Lines and Antibody Analysis

Neuroblastoma cell lines used were CLB-BAR (amplified MYCN/ALK, Δ4-11), CLB-GE (amplified MYCN/ALK, ALK-F1174V), CLB-PE (amplified MYCN, WT ALK) and IMR-32 (amplified MYCN, WT ALK) and PC12 cells were cultured and grown as previously reported [7 (link), 35 (link)]. Primary antibodies employed for immunoblotting were: phospho-ALK (Y1604), phospho-AKT (S473), MYCN, phospho-ERK1/2 (1:2000), phospho-ERK5 (1:1000), ERK5, panERK, Actin (1:5000) from Cell Signaling Technology (Danvers, MA). Monoclonal antibody 135 (anti-ALK) was produced in the Hallberg laboratory against the extracellular domain of ALK [38 (link)]. Horseradish peroxidase conjugated secondary antibodies; goat anti-rabbit IgG and goat anti-mouse, IgG (1:5000) were obtained from Thermo Scientific. Brigatinib, was obtained from Ariad Pharmaceuticals and crizotinib was from Haoyuan Chemexpress Co., Limited, Shanghai.