Pretreatment of FA IL-3Rα⁺ blasts with the IL-3Rα–neutralizing antibody inhibits IL-3–mediated proliferation and STAT5 activation. (A) Human CD45⁺IL-3Rα⁺ cells from the bone marrow of FA leukemic recipient mice were isolated by FACS and cultured in the IL-3–neutralizing Ab 7G3 (100nM) or the isotype-matched antibody IgG2a (100nM) for 6 hours in the absence or presence of IL-3 (10 ng/mL), followed by BrdU incorporation assay. (B) Human CD45⁺IL-3Rα⁺ cells from the bone marrow of FA leukemic recipient mice were isolated by FACS and cultured in the IL-3–neutralizing Ab 7G3 (100nM) or the isotype-matched antibody IgG2a (100nM) for 6 hours in the absence or presence of GM-CSF (5 ng/mL), followed by BrdU incorporation assay. P value indicates comparison of difference between 7G3-treated and -untreated groups. (C) Cells described in panel B were cultured in the IL-3–neutralizing Ab 7G3 (100nM) or the isotype-matched antibody IgG2a (100nM) for 6 hours. Apoptosis was determined by Annexin V/7AAD double staining, followed by flow cytometric analysis. (D) Whole-cell extracts were prepared from the cells described in panel A, followed by immunoprecipitation with the anti-JAK2 antibody. The immune complexes were resolved on 7.5% SDS-PAGE, transferred to nylon membrane, and probed with anti-JAK2 or anti-phosphotyrosine mAb 4G10. (E) Whole-cell extracts from cells described in panel A were subjected to SDS-PAGE and immunoblotted with antibodies specific for phosphor-STAT5a, total STAT5a, or β-actin.