NPM-ALK binds and phosphorylates p130Cas. (A) The 293 T-Rex Tet-On cells were cultured with tetracycline for 24 hours; total cell lysate was immunoprecipitated (IP) with anti-ALK monoclonal antibody and blotted with the indicated antibodies by WB. NPM-ALK coprecipitates with p130Cas, paxillin, and Src. (B) NPM-ALK coprecipitates both with p130Cas (arrow) and with Cas-L (arrowhead) in lymphoid cell lines. Total lysates from ALK-positive (DHL, TS) and ALK-negative (CEM, K562) cell lines were immunoprecipitated with anti-ALK monoclonal antibody and blotted with the indicated antibodies. (C) In ALK-positive lymphoid cells, both p130Cas and Cas-L are phosphorylated. Total lysates were immunoprecipitated with anti-p130Cas monoclonal antibody and blotted with the anti-PY20 monoclonal antibody. (D) The adaptor p130Cas is phosphorylated in the presence of NPM-ALK. The 293 T-Rex Tet-On cells expressing NPM-ALK were cultured with tetracycline for 24 hours; cell lysate was immunoprecipitated with anti-PY20 monoclonal antibody and blotted with the indicated antibodies. (E) The binding and the phosphorylation of p130Cas depends on NPM-ALK kinase activity. The 293 T-Rex Tet-On cells expressing the active form of NPM-ALK or the kinase-dead control (NPM-ALK^K210R) were cultured with tetracycline for 24 hours; cell lysates were immunoprecipitated with anti-ALK monoclonal antibody and blotted with the indicated antibodies. (F-G) The phosphorylation of p130Cas does not depend on the binding with the NPM portion of NPM-ALK. The 293 T cells were transfected with Pallino p130Cas alone or in combination with Pallino NPM-ALK or Pallino ATIC-ALK. Samples were collected 48 hours after transfection and total lysates were immunoprecipitated and blotted with the indicated antibodies.