Identification and analysis of mutated ALK in neuroblastoma

Genome-wide comparative genomic hybridization analysis and subsequent direct sequencing of neuroblastoma cell lines and primary tumour DNAs allowed us to identify somatic mutations of the ALK-kinase domain. Germline mutations were observed in two neuroblastoma families indicating that ALK is a neuroblastoma predisposition gene. Mutated ALK proteins were over-expressed, hyperphosphorylated and exhibited constitutive kinase activity. The knock-down of ALK expression in ALK-mutated cells, but also in cell lines over-expressing a wild-type ALK, led to a dramatic decrease of cell proliferation. Here, we provide information about the reagents used in this study and describe the RNA interference method, immunoprecipitation protocol and in vitro kinase assay