NPM-ALK binds p130Cas through the SH2 domain of Grb2. (A) NPM-ALK binds Grb2 in lymphoid cell lines. Total lysates were immunoprecipitated with anti-ALK monoclonal antibody and blotted with the indicated antibodies. (B-C) The 293 T cells were transfected with Pallino NPM-ALK, Pallino p130Cas, pRK5 Grb2, and the dominant-negative Grb2 constructs pRK5 P49L (mutated in the SH3 domain) or pRK5 R86K (mutated in the SH2 domain) as indicated. Samples were collected 48 hours after transfection and total lysates were immunoprecipitated with anti-Grb2 polyclonal antibody and blotted with the indicated antibodies. Grb2 R86K was able to disrupt the binding of p130Cas to NPM-ALK (B) as well as p130Cas phosphorylation (C).