NPM-ALK dephosphorylation by SHP1 in ALCL and NIH3T3 cells. (A) Karpas 299 cells (6 × 10⁶) were transfected with 200 nM control siRNA (lane 1) or 200 nM SHP1 siRNA (lane 2). After 48 hours of cell culture, 10⁷ cells were lysed and immunoblotting with anti-SHP1 antibody was performed from 25 μL of each lysate (top panel). NPM-ALK was immunoprecipitated with the ALK1 antibody and immune complexes were analyzed by immunoblotting with 4G10 antiphosphotyrosine antibody (top left panel). To assess the quantity of immunoprecipitated NPM-ALK, membrane was reprobed with the ALKc antibody (bottom left panel). Forty-eight hours after transfection with control siRNA (lane 1) or SHP1 siRNA (lane 2), STAT3 immunoprecipitations were performed and the immune complexes were submitted to Western blot using phospho-STAT3 (Tyr705) antibody (top right panel). The membrane was reprobed with anti-STAT3 antibody to assess STAT3 loading (bottom right panel). (B) Karpas 299 and SU-DHL1 cells were stained with anti-SHP1 or phospho-STAT3 antibodies and were observed by confocal microscope as described in “Materials and methods.” A strong nuclear staining for STAT3 is seen in SHP1-negative SU-DHL1 cells, whereas SHP1-positive Karpas cells show a weak doubtful nuclear staining. Total magnification is 63 ×. (C) NIH3T3 fibroblasts stably expressing NPM-ALK were transfected with 5 μg of pcDNA3 (lane 1) or pcDNA3-SHP1 (lane 2). After 15 hours of culture, SHP1 overexpression was detected from lysates of transfected NIH3T3 fibroblasts by anti-SHP1 Western blot (left panel). Transfected cells (5 × 10⁶) were subjected to NPM-ALK immunoprecipitation and immune complexes were analyzed as in panel A. Results reported in this figure are representative of 3 independent experiments.