Induction of tyrosine phosphorylation of JAK2 after stimulation of UT-7/GM cells with GM-CSF, EPO, TPO, or their combinations. GM-CSF was removed from UT-7/GM cells overnight. The cells were then stimulated with medium alone, TPO (100 ng/mL), EPO (10 U/mL), GM-CSF (10 ng/mL), or their combinations for 5 minutes before solubilization. Cell lysates were immunoprecipitated with Protein G Sepharose-conjugated anti-JAK2 serum. Immunoprecipitates were eluted with buffer containing SDS and resolved by 7.5% SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane. (A) Immunoblotting with anti-P-Tyr antibody (4G10). (B) Immunoblotting with anti-JAK2 serum. The blot was reprobed with anti-JAK2 serum to confirm equal loading of the samples. (C) Densitometric analysis of tyrosine phosphorylated JAK2 proteins. (D) The effects of EPO, TPO, GM-CSF, or their combinations on the proliferation of UT-7/GM cells. Proliferative responses of UT-7/GM cells to cytokines were analyzed by the MTT assay (see Materials and Methods). Cells were plated at a density of 10⁴/well in IMDM supplemented with 5% FCS and cultured with TPO (100 ng/mL), GM-CSF (10 ng/mL), EPO (10 U/mL), or their combinations. MTT reduction was measured after 3 days of culture. The values represent the mean ± SD from triplicate cultures.