Molecular characterization of NPM-ALK Tg mice.

(A) Expression and constitutive activation of NPM-AK in Tg mice. Thymocytes from Tg and WT mice were lysed and immunoprecipitated with anti-ALK Ab as described in “Materials and methods.” Western blot with anti-ALK revealed the presence of the protein in Tg but not WT mice (left panel). NPM-ALK protein was constitutively phosphorylated in Tg mice as revealed by the antiphosphotyrosine Ab (right panel). (B) NPM-ALK protein expressed in murine T cells coprecipitates with Shc, IRS-1, Grb-2, and PI3K. Lysed from ALK⁺samples were immunoprecipitated with anti-ALK (upper panel) or with anti–Grb-2 or anti-PI3K (lower panel). Immunocomplexes were gel electrophoresed and, after transfer, incubated with the indicated antibodies. Direct Western blotting were also performed as indicated. All data are representative of at least 3 different experiments. (C) NPM-ALK Tg mice activate Stat3. Proteins extracted from Tg and WT thymocytes were immunoprecipitated with anti-Stat3 Ab and loaded onto a SDS-PAGE gel. Stat3 protein was similarly immunoprecipitated from both Tg and WT thymocytes. Antiphosphotyrosine Ab revealed the presence of higher levels of activated Stat3 in NPM-ALK Tg mice. (D) NPM-ALK Tg mice activate Jak3. Jak-family of proteins were immunoprecipitated from Tg and WT mice and detected with antiphosphotyrosine Ab. Only Jak3 was constitutively phosphorylated in Tg but not in WT mice. The Jak-family of proteins were equally immunoprecipitated in Tg and WT mice.