DEP-1 is inactivated by ROS in FLT3 ITD–expressing cells. (A) Rescue of DEP-1 activity by coexpression of catalase (CAT). The experiment was performed as in Figure 1E (means of normalized values for 3 separate experiments ± SD, significance tested by t test). (B) Partial recovery of DEP-1 activity by DTT treatment. DEP-1 was immunoprecipitated from HEK293 cells, which were transiently transfected as indicated. The immunoprecipitates were treated with 5mM DTT at room temperature for 10 minutes, or mock-incubated without DTT as indicated, then washed 3 times with PTP assay buffer, and the activity was subsequently assayed using the phosphopeptide substrate (values corrected for nonspecific precipitation with IgG controls; means of normalized values for 3 separate experiments ± SD, significance tested by t test). (C), FLT3 ITD causes ROS-mediated modification of the DEP-1 catalytic cysteine. HEK293 cells were transiently transfected with the indicated expression constructs, and lysed in the presence of biotinylated iodoacetate (EZ-link idoacetyl-PEG₂-biotin). DEP-1 was immunoprecipitated, and incorporation of biotin was assessed by immunoblotting. The blot was reprobed for comparable amounts of DEP-1. A representative experiment (top panel) and quantification of 3 experiments (means of normalized values for the ratio of signals for biotinylated DEP-1 and total DEP-1 ± SD, significance tested by t test, bottom panel) is shown. (D-E) DPI reactivates DEP-1 in FLT3 ITD–expressing cells. HEK293 cells were transiently transfected with the indicated expression constructs, and were left untreated or were treated with 0.5μM DPI for 6 hours before lysis. Thereafter, DEP-1 activity was assessed in immunoprecipitates (D; values corrected for nonspecific precipitation with IgG controls; means of normalized values for 3 separate experiments ± SD, significance tested by t test), or phosphorylation of FLT3 ITD was analyzed by immunoblotting (E). Top panel: representative experiment. Bottom panel: quantification of 3 separate exeriments (means of normalized values for the ratio of signals for phosphorylated FLT3 and total FLT3 ± SD, significance tested by t test). (F) VAS2870, a NADPH-oxidase inhibitor, reactivates DEP-1 in FLT3 ITD–expressing cells. FLT3 ITD–expressing 32D cells were treated with 50μM VAS2870 for 5 hours, thereafter ROS levels were determined using carboxy-H₂DFFDA and the DEP-1 activity was assayed measuring dephosphorylation of a phosphotyrosine-containing phosphopeptide with the malachite green assay in DEP-1 immunoprecipitates (means of normalized values for 3 separate experiments ± SD, significance tested by t test). Cell lysis, immunoprecipitation, and activity assay were performed in an anaerobic chamber.