Association between SHP1 and NPM-ALK. (A) Pull-down experiments were performed with 5 μg of GST, GST-SHP1 C/S, or GST-SHP1 WT fusion proteins incubated with total cell extracts from 10⁷ Karpas 299 cells (lanes 1, 3, and 4) or from 10⁷ FE-PD cells (lane 2) pretreated with orthovanadate (500 μM for 1 h). Association of NPM-ALK with these fusion proteins was analyzed by immunoblotting with the ALKc antibody (top panel). The input recombinant proteins were detected with anti-GST antibody (bottom panel). Results are representative of 3 independent experiments. (B) Whole-cell extracts from 10⁷ Cost or Karpas 299 cells were immunoprecipitated with anti-SHP1 antibody and immunoblotted with the ALKc antibody (top middle panel). To assess the quantity of immunoprecipitated SHP1, membranes were reprobed with anti-SHP1 antibody (bottom middle panel). NPM-ALK immunoprecipitates (using the ALK1 antibody) were subjected to immunoblotting with anti-SHP1 antibody (top right panel). Membranes were then reprobed with the ALKc antibody (bottom right panel). Immunoprecipitations were performed without addition of antibody as negative control (left panels). Data are representative of 5 independent experiments.