Dysfunction of DEP-1 in FLT3 ITD–expressing cells. 32D cells, either expressing WT FLT3 (A) or FLT3 ITD (B), which were stably transduced with nontargeting (control) or DEP-1 targeting shRNA (as indicated), or MV4-11 cells, transiently transfected with a DEP-1–targeting siRNA (C) were starved and stimulated with FLT3 ligand (FL) for the indicated time periods. FLT3 phosphorylation was assessed by immunoblotting in cell lysates with antibodies recognizing phosphorylated FLT3 (pFLT3, pY589, or pY591). Numbers under the blot indicate relative phosphorylation of FLT3, normalized to FLT3 amounts and to the value at 2.5 minutes stimulation in the control sh/siRNA-treated cells (1.0). Blots in the right panel reveal the DEP-1 knockdown in the used 32D cell pools, which were assayed in parallel to the signaling experiments. Note that blot sections are from the same membranes with identical exposure and image processing, but were rearranged for better clarity. DEP-1 activity was assayed in stably transfected 32D cells (D) or transiently transfected HEK293 cells (E) by measuring dephosphorylation of a phosphotyrosine-containing phosphopeptide with the malachite green assay in DEP-1 immunoprecipitates (values corrected for nonspecific precipitation with IgG controls; means of normalized values for 3 separate experiments ± standard deviation (SD), significance tested by t test). Cell lysis, immunoprecipitation, and activity assay were performed in an anaerobic chamber. Bottom panels verify equal DEP-1 protein levels.