PI 3-kinase was constitutively activated in HCD57 and HCD57-SREI cells. (A) Analysis of PI 3-kinase activity in HCD57 and HCD57-SREI cells. (Top panel, A) 3 × 106 HCD57 (lanes 1 and 2) and HCD57-SREI (lanes 3 and 4) cells were deprived of EPO overnight and treated with 10 U/mL EPO (lanes 2 and 4) for 5 minutes or left untreated (lanes 1 and 3). Cells were lysed and immunoprecipitated by anti-p85 subunit of PI 3-kinase antibody, and the precipitates were assayed for PI3-kinase activity as described in Materials and Methods. The positions of the PI 3-phosphate product (PI3-P) and the origin are indicated. (B) Inhibition of in vitro PI3-kinase activity by LY294002. (Top panel, B) HCD57 cells were deprived of EPO overnight and treated with EPO for 5 minutes. Immunoprecipitates were prepared from HCD-57 cells as described previously. PI 3-kinase assay was performed in the presence of various concentrations of LY294002 (lanes 5 through 8) as indicated. (Bottom panels, A and B) PI 3-kinase activities were quantified and the results were expressed as fold activity compared to the activity of HCD57 cells not treated with EPO. Data are means ± SD from three independent experiments. (C) Analysis of PI 3-kinase activity in different immunoprecipitates. Cell lysates were prepared from HCD-57 cells cultured in normal medium and immunoprecipitated by anti-p85 subunit of PI 3-kinase (lane 9), anti-JAK2 (lane 10), anti-STAT5 (lane 11), or anti-Vav (lane 12) antibodies. The precipitates were assayed for PI 3-kinase activity as described in Materials and Methods. The positions of the PI3-phosphate product (PI3-P) and the origin are indicated.
