Activity of other protein-tyrosine phosphatases in FLT3 ITD–expressing cells. (A) Oxidation of cytoplasmic PTPs in 32D cells was assessed by the modified in-gel assay. Lysates of parental 32D cells, or 32D cells expressing WT FLT3 or FLT3 ITD (as indicated) were prepared in presence of the alkylant iodoacetic acid (IAA). For positive controls, parental 32D cells were pretreated with 1mM H₂O₂ for 10 minutes, and then lysed with IAA, or were lysed without IAA, as indicated. Lysate aliquots were separated in an SDS-PAGE gel containing [³²P]phosphotyrosine-poly(Glu₄Tyr), and the gels were processed for PTP renaturation, dried, and subjected to autoradiography. Subsequent to lysis in presence of IAA, only reversibly oxidized PTPs can be detected as white bands. Molecular masses are indicated. (B-C) The activity of the transmembrane PTP CD45 or PTP1B was also assessed in absence or presence of FLT3 ITD. (B) For PTP1B assays, endogenous PTP1B was immunoprecipitated from HEK293 cells, transiently transfected with control vector (VC) or an expression construct for FLT3 ITD. (C) For CD45 measurements, endogenous CD45 was immunoprecipitated from 32D cells stably expressing FLT3 ITD or parental 32D cells. Activity was measured in an anaerobic compartment by malachite green assays (values corrected for nonspecific precipitation with IgG controls; means of normalized values for 3 separate experiments ± SD, significance tested by t test). (D) Possible oxidation of the lipid phosphatase PTEN in presence of FLT3 ITD was assessed. HEK293 cells were transfected as indicated, and lysed in presence of the alkylating agents N-ethylmaleimide (NEM) and IAA in an anaerobic chamber. For positive control, one set of cells was also treated with 0.2mM H₂O₂ for 10 minutes before lysis. Proteins were precipitated with trichloroacetic acid to remove the alkylants, redissolved, subjected to reducing treatment, and subsequently reacted with biotin-conjugated maleimide. Biotinylated proteins were enriched with streptavidin-beads and analyzed by SDS-PAGE and immunoblotting. PTEN detected in this fraction was designated biot-PTEN. Aliquots of fractions before the final affinity step were also analyzed as input controls (lowest panel). (E-F) HEK293 cells, which were transiently transfected as indicated (E), and MOLM-13 cells or MV4-11 cells expressing endogenously FLT3 ITD (F) were lysed in presence of 40mM NEM in an anaerobic chamber. Samples were fractionated by nonreducing SDS-PAGE. Oxidation of PTEN caused the formation of a band which migrates faster (designated ox) than the reduced form (red). For an additional specificity control, a PTEN cysteine-to-serine mutant (CS), which cannot become oxidized, was also analyzed. (G) MOLM-13 cells were serum-starved and treated with 100μM bV(phen), a general PTP inhibitor, for 15 minutes. Subsequently, cells were stimulated with FLT3 ligand (FL) for different time as indicated, lysed, and FLT3 was immunoprecipitated. Phosphorylation was analyzed by immunoblotting. Numbers under the blot indicate relative phosphorylation of the mature (top) FLT3 band, normalized to total FLT3 and phosphorylation at 2.5 minutes after FL-stimulation (1.0).