The phosphorylation of p130Cas by NPM-ALK is independent from Src tyrosine kinase activity. (A) NPM-ALK coprecipitates with Src in lymphoid cells. Total lysates from ALK-positive (Karpas, DHL, TS) and ALK-negative (CEM, Namalwa) cell lines were immunoprecipitated with anti-ALK monoclonal antibody and blotted with anti-Src polyclonal antibody. (B) The NPM-ALK–mediated phosphorylation of p130Cas is Src independent. Syf triple knock-out fibroblasts were cotransfected with Pallino p130Cas and Pallino NPM-ALK or Pallino NPM-ALK^K210R as a control. Samples were collected 48 hours after transfection and total lysates were immunoprecipitated with anti-PY20 monoclonal antibody and blotted with the indicated antibodies. (C) Src inhibition does not limit p130Cas phosphorylation. MEF NIH3T3 cells were cotransfected with Pallino p130Cas and Pallino NPM-ALK or Pallino NPM-ALK^K210R as a control. Forty-eight hours after transfection, samples were cultivated in the presence of 30 μM PP2 for 1 hour and then collected. The adaptor p130Cas phosphorylation levels were detected by immunoprecipitation with anti-p130Cas monoclonal antibody followed by blotting with anti-PY20 monoclonal antibody. (D) ALK-positive (DHL and TS) and ALK-negative (K562) cells were cultivated in the presence of 30 μM PP2 for 1 hour and then collected. The adaptor p130Cas phosphorylation levels were detected by immunoprecipitation with anti-p130Cas monoclonal antibody followed by blotting with anti-PY20 monoclonal antibody.