NPM-ALK and p85 form a complex in vivo via the C-terminal SH2 domain of p85.

The 2 × 10⁷ Ba/F3 cells stably transfected with NPM-ALK (A) or 2 × 10⁷ Karpas299 cells (B) or 2 × 10⁷SUP-M2 cells (C) were subjected to immunoprecipitations and immunoblotting with the antibodies indicated. Association of NPM-ALK with p85 is demonstrated by coprecipitation of p85 with an NPM-ALK antibody (A and B, left panel, right lane; C, left panel, middle lane) and by coprecipitation of NPM-ALK with anti-p85 antibody (A, B, C, right panel, right lane). A rabbit antimouse antibody (RαM) served as a negative control for the immunoprecipitation. (D) Left panel: 1 × 10⁷ Karpas299 cells were lysed, then incubated with the C- or N-terminal SH2 domain GST-fusion proteins for 1 hour at 4°C. The bound (B) and the flow-through (FT) fractions were collected with glutathione-Sepharose, subjected to 7.5% SDS-PAGE, and analyzed by anti-ALK immunoblotting. Right panel: Lysates of 1 × 10⁷ Ba/F3 cells cultured with and without IL-3 for 2 hours as indicated were incubated with the N-terminal p85 SH2 domain or GST as control for 1 hour at 4°C and the bound fractions were collected with glutathione-Sepharose. Input and bound fractions were subjected to 7.5% SDS-PAGE, and analyzed by antiphosphotyrosine immunoblotting.