ALK⁺ ALCL-cell lines express IL-9Rα. (A) RT-PCR studies show the presence of IL-9Rα mRNA in all ALK⁺ ALCL cells. The Hodgkin lymphoma-cell line L1236 was used as positive control and the colon carcinoma HT29-cell line as negative control. β-Actin shows equal loading. (B) Immunohistochemical staining of paraffin-embedded tissue sections from cell blocks confirmed the expression of IL-9Rα in all the ALK⁺ ALCL-cell lines. Similar to RT-PCR studies, L1236 and HT29 cells were used as positive and negative controls, respectively (original magnification × 200). (C) Confocal microscopy after immunofluorescence staining of cytospin slides with antibodies directed against IL-9Rα (bottom panel) or control IgG (top panel) demonstrates the expression of IL-9Rα in the ALK⁺ ALCL-cell lines, Karpas 299, SU-DHL-1, and SUP-M2 (original magnification × 600). (D) Immunoprecipitation and Western blotting show the physical association between γc and pJak3 in Karpas 299 cells. As shown in the right panel, immunoprecipitation was first performed on the cell lysate using anti-γc antibody followed by Western blotting using anti-pJak3 antibody. The left panel demonstrates a simultaneous control study where the lysate was analyzed only by Western blotting using an anti-pJak3 antibody. pJak3 bands are present at the expected molecular weight of 118 kDa in the 2 panels. Similar findings were noted when anti-Jak3 antibody was used instead of anti-pJak3 antibody (data not shown).