PI 3-kinase activity associates with the EPOR and IRS-2. (A) 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-EPOR antiserum, and the precipitates were assayed for PI3-kinase activity as described in Materials and Methods. The positions of the PI3-phosphate product (PI3-P) and the origin are indicated. (B) HCD57 (lanes 5 and 6) and HCD57-SREI (lanes 7 and 8) cells were deprived of EPO overnight and treated with 10 U/mL EPO (lanes 6 and 8) for 5 minutes or left untreated (lanes 5 and 7). Cells were lysed and immunoprecipitated by anti–IRS-2 antibody, and the precipitates were assayed for PI3-kinase activity as described in Materials and Methods. The positions of the PI3-phosphate product (PI3-P) and the origin are indicated.