NPM-ALK tyrosine phosphorylation in Ba/F3 cells and ALCL-derived cell lines. (A) Ba/F3 neo and Ba/F3 N/A cells were deprived of serum for 24 hours, prior to serum restimulation for various times before cell lysis and immunoblot analysis with an antiphosphotyrosine antibody 4G10. The nitrocellulose membrane was stripped and reprobed with anti-ALK and antiactin antibodies to assess NPM-ALK expression and protein loading, respectively (middle panel). Band intensity was semiquantified by densitometric scanning of the film (actin: white; NPM-ALK: black). Protein extracts from 5 × 10⁶ cells were loaded in each lane. (B) As a control, Ba/F3 N/A cells cultured with or without IL3 were used for immunoblotting with the 4G10 antibody. (C) For NPM-ALK immunodepletion experiments, Ba/F3 N/A cells were harvested 6 hours after serum restimulation and subjected to immunoprecipitation with or without the ALK1 antibody (1: supernatant; 2: beads) followed by immunoblotting with the 4G10 antibody. (D) NPM-ALK–negative (FEPD) and –positive (COST, KARPAS, and SU-DHL1) ALCL cell lines were maintained in normal culture conditions as described in “Materials and methods” prior to cell lysis and immunoblot analysis with the 4G10 antibody (upper panel) or with the anti-ALKc antibody (middle panel). The nitrocellulose membrane was stripped and reprobed with antiactin antibody to assess protein loading (lower panel). From cell protein extracts, 60 μg was loaded in each lane. Data shown in this figure are representative of 3 to 4 independent experiments.