Overactivation of JAK2/STAT5 in FA IL-3Rα–positive blasts in response to IL-3. (A) Human CD45⁺IL-3Rα⁺ cells from the bone marrow of FA leukemic recipient mice were isolated by FACS, cultured in the presence or absence of IL-3 (10 ng/mL) for 15 minutes, and whole-cell extracts were prepared. Total proteins (0.5-1 mg) for each indicated sample were subjected to immunoprecipitation with anti-JAK2 antibody. The immune complexes were resolved on 7.5% SDS-PAGE, transferred to nylon membrane, and probed with the anti-phosphotyrosine mAb 4G10 or an anti-JAK2 antibody. (B) Direct Western blot for STAT5a. Whole-cell extracts (∼ 100 μg of total proteins for each indicated sample) from the cells described in panel A were subjected to SDS-PAGE and immunoblotted with antibodies specific for phosphor-STAT5a, total STAT5a, or β-actin.