FIGURE 1.
Phosphorylation of Grb2 in NPM-ALK-expressing cells. A, Grb2 low mobility (m) band and binding do not depend on the type or localization of the ALK fusion proteins. Anti-ALK immunoprecipitations were performed on HEK-293T cells transiently transfected with the indicated ALK full-length and ALK fusion proteins with or without Grb2. Western blots of total lysates and anti-ALK-immunoprecipitated (IP) proteins were blotted with the indicated antibodies. B, HEK-293T cells transiently transfected with NPM-ALK or the kinase-dead mutant were immunoprecipitated with anti-ALK antibodies and blotted with ALK and Grb2 antibodies. C, Grb2 low mobility band is detected only in the presence of the active NPM-ALK but not the kinase-dead NPM-ALKK210R in HEK-293T cells transfected with Grb2 and NPM-ALK or NPM-ALKK210R and blotted with the indicated antibodies. D, Grb2 lower mobility band is detected in ALCL cells. The TS and SU-DHL-1 cell lines were transduced with an inducible anti-ALK shRNA lentivirus (TTA ALK) as described under “Material and Methods.” Cells were cultivated in presence of 1 μg/ml doxycycline (DOXY) for 72 h to knock down NPM-ALK expression, and cell lysates were analyzed by Western blot. E, HEK-293T co-transfected with NPM-ALK and Grb2 were treated with CEP-14083 inhibitor (300 μm) 24 h after transfection for the indicated time points. The effect on NPM-ALK, Grb2, and Erk1/2 phosphorylation was followed over time on total cell extracts by Western blot (nt, not transfected). F, NPM-ALK expression was induced in HEK-293T-Rex by the addiction of 1 μg/ml tetracycline (Tet) to the culture for 24 h. Cell lysates were incubated with or without λPPase. Western blots were blotted with the indicated antibodies.