ALK oncogene expression in spleen, lymph node, and blood of TPM3-ALK and NPM-ALK double-transgenic mice. Immunohistochemical analysis of spleen (A-C, original magnification ×1000), lymph node (D, original magnification ×640; E-F, original magnification ×1000), blood lymphoid cells (G-I, original magnification ×1000) from control age-matched tTA (A,D,G), TPM3-ALK ON (tTA/pBIL-TA) (B,E,H), and NPM-ALK ON (tTA/pBIL-NA) (C,F,I) double-transgenic mice. Sections were stained with the rabbit anti-ALK antibody, and nuclei were counterstained with hematoxylin. NPM-ALK–positive tumor cells show a cytoplasmic and nuclear and nucleolar staining (C,F,I) in comparison with TPM3-ALK–positive tumor cells, which harbored a cytoplasmic and membrane staining (B,E,H). The staining intensity varies from cell to cell. Scattered ALK-positive lymphoma cells are observed in lymphatic sinuses (E-F). Confocal microscopy analysis shows the restricted cytoplasmic staining in TPM3-ALK (K) and the cytoplasmic and nucleolar staining in NPM-ALK (L) double-transgenic mice. Sections were stained with the mouse anti-ALK antibody, and nuclei were counterstained with TO-PRO-3 iodide. (M) Western blotting analysis of TPM3-ALK and NPM-ALK expressions. Protein lysates (100 μg) extracted from lymph nodes (LN) and spleens (S) of tTA/pBIL-TA and tTA/pBIL-NA double-transgenic mice; tTA, pBIL-TA, and pBIL-NA control transgenic mice; and positive control Karpas cell line overexpressing NPM-ALK (lane C+) were subjected to Western blotting analysis with anti-ALKc, antiphosphotyrosine, and anti–β-actin antibodies. (N) Flow cytometric analysis of ALK expression in lymph node cells from NPM-ALK double-transgenic mice. The histograms show ALK expression (red line) and isotype control (black line). Confocal microscopy (O) of lymphoma cells shows the heterogeneity of ALK expression as seen with flow cytometric analysis.