Activation of pp60^c-src in NPM-ALK–expressing cells. (A) Ba/F3 neo and Ba/F3 N/A cells were subjected to the same serum deprivation/restimulation time course (24-hour serum withdrawal followed by 0-, 6-, and 24-hour serum readdition). Immunoblotting analysis of protein extracts (from 5 × 10⁵ cells) was performed using anti-Src pTyr418 (signature of maximal activation of all Src-kinases), Src pTyr215 antibodies (specific for the activated form of pp60^c-src), and anti-Src2 antibody (to visualize Src-family members). (B) Activation of Src-kinases was also revealed using the same antibodies in 4 ALCL-derived cell lines: FEPD (NPM-ALK negative), COST, KARPAS, and SU-DHL1 (NPM-ALK positive). Loaded per lane was 60 μg cell protein extracts. (C) NPM-ALK was immunoprecipitated (from 5 × 10⁶ cells) with the ALK1 antibody followed by immunoblotting using the anti-Src pTyr418 antibody to analyze the activation state of the associated Src-kinases. The same nitrocellulose was stripped and reprobed using anti-Src2 antibody to visualize Src-family members. In panels A-B, proteins were separated on a 7.5% SDS-PAGE, whereas a 12% SDS-PAGE was used in panel C to achieve a higher resolution. Data shown are representative of 3 independent experiments with similar results.