The phosphorylation of c-Kit or IL-3R/β in Lin–CD34– and Lin–CD34+EML cells in response to different cytokines. (A) Total cell lysates of Lin–CD34+ and Lin–CD34– EML cells were separated by SDS/PAGE and transferred to an Immobion-P membrane. The membrane was probed with anti-c-Kit antibody, stripped, and reprobed with anti-β-tubulin antibody. IB, immunoblotting. (B and C) Lin–CD34+ (B) and Lin–CD34– (C) EML cells were starved and treated with no stimulation, SCF (100 ng/ml), IL-3 (5 ng/ml), or the combination IL-3 (5 ng/ml) and SCF (100 ng/ml) for 10 min. Cells were harvested, and c-Kit was immunopreciptated (IP) with c-Kit antibody (M14). The immunopreciptates were analyzed by SDS/PAGE, followed by immunoblotting with the indicated antibodies. After being hybridized with antiphosphotyrosine Y719-specific c-Kit antibody, the same membrane was stripped and reprobed with the general antiphosphotyrosine antibody PY100 and anti-c-Kit antibody. (D) Lin–CD34– EML cells were starved and treated with no stimulation, SCF (100 ng/ml), IL-3 (5 ng/ml), or the combination IL-3 (5 ng/ml) and SCF (100 ng/ml) for 10 min. Cells were harvested, and IL-3R/β was immunopreciptated with anti-IL-3R/β antibody (K17). The immunopreciptates was analyzed by SDS/PAGE, followed by immunoblotting with antiphosphotyrosine antibody PY100. The membrane was then striped and reprobed with IL-3R/β antibody.