In vitro kinase assay

The Universal Tyrosine Kinase Assay kit (Takara) was used to measure kinase activity according to the manufacturer’s instructions. Briefly, NIH 3T3 cells stably expressing WT ALK or ALK CSMs were harvested in extraction buffer, lysates incubated on ice for 20 min, centrifuged at 10,000 g for 10 min at 4°C and the supernatants pre-cleared with 50 µL protein G agarose beads for 30 min at 4°C under rotation. 10 µL ALK antibody was then added to the supernatant and incubated at 4°C under rotation for 4 hr. 30 µL protein G agarose beads were then added to the supernatant and incubated at 4°C under rotation for 2 hr. The immunoprecipitated material was then washed with PBS and used in the kinase assay.