NPM-ALK tyrosine phosphorylation and SHP1 phosphatase activity in Cost and Karpas 299 ALK-positive ALCL-derived cell lines. (A) Western blotting analysis of tyrosine phosphorylation status with the 4G10 antiphosphotyrosine antibody in total cell extracts from Cost and Karpas 299 cell lines. (B) NPM-ALK immunoprecipitation (IP) from 10⁷ cells with the ALK1 antibody followed by immunoblotting (IB) with the 4G10 antiphosphotyrosine antibody (i). Nitrocellulose membrane was stripped and reprobed with the ALKc antibody to assess NPM-ALK loading (ii). (C) Detection of SHP1 by Western blotting performed on Cost and Karpas 299 cell lysates showing a stronger expression of SHP1 in Karpas cells compared with Cost cells. (D) For the SHP1 phosphatase activity assay, anti-SHP1 immunoprecipitates from Cost and Karpas cell lines were incubated for 30 minutes at 30°C with P-NPP as a substrate. The OD of supernatants was measured at 410 nm and immune complexes were submitted to immunoblotting with an anti-SHP1 antibody. SHP1 phosphatase activities, expressed in OD measurements, were related to the same quantity of SHP1 protein (mean ± SD of 3 independent experiments; statistically significant difference [Student t test] was observed, ** P < .01). (E) SHP1 immunoprecipitates from Cost and Karpas cell lines were submitted to an in-gel phosphatase assay as described in “Materials and methods.” The higher level of SHP1 phosphatase activity in Karpas compared with Cost cells is in agreement with the results shown in panels C and D. Data shown in this figure are representative of 3 independent experiments.