NPM-ALK dephosphorylation by SHP1 in vitro. (A) Antiphosphotyrosine immunoprecipitates were performed from 107 Karpas 299 cells (right panel) and FE-PD cells (left panel) pretreated with orthovanadate (500 μM for 1 hour). Immunoprecipitated phosphoproteins were incubated in phosphatase buffer with GST-SHP1 WT or GST-SHP1 C/S fusion proteins for 1 hour at 30°C and submitted to antiphosphotyrosine immunoblotting (top panels). The membrane was reprobed with the ALKc antibody to assess NPM-ALK loading (bottom panel). Data are representative of 3 independent experiments. (B) NPM-ALK immunoprecipitates from 107 Karpas cells, pretreated with orthovanadate, were incubated in the same PTP assay conditions (see above), with or without SHP1 immunoprecipitates performed on untreated Karpas 299 cells. Proteins in immune complexes were separated by SDS-PAGE and immunoblotted with antiphosphotyrosine antibody (top panel). Note that an equal amount of NPM-ALK was immunoprecipitated in each experiment (bottom panel). The data are representative of 2 independent experiments.
