![Fig. 3. In vitro kinase assay revealed that AKT kinase is activated by EPO stimulation. / (A) EPO was removed from UT-7/EPO cells for 24 hours. Then the cells were stimulated with EPO (10 U/mL) for 10 minutes. After solubilization, cell lysates were immunoprecipitated with protein G-conjugated anti-Akt antibody. Then immunoprecipitates were subjected to an in vitro kinase assay. The kinase assays were carried out in the presence of 10 μCi of [γ-32P]ATP (3000 Ci/mmol) using histone H2B as a substrate (0.1 mg/mL). The reactions were incubated at 25°C for 30 minutes. Reaction products were resolved by 15% SDS-PAGE and visualized by autoradiography. (B) EPO was removed from UT-7/EPO cells for 24 hours, then the cells were incubated with 50 μmol/L LY294002 for 45 minutes and stimulated with EPO (10 U/mL) for 10 minutes. Cells were lysed and immunoprecipitated with anti-Akt antibody. Then immunoprecipitates were subjected to an in vitro kinase assay. The kinase assays were carried out in the presence of 200 μmol/L ATP using GSK-3 as a substrate (0.025 mg/mL) . The reactions were incubated at 30°C for 30 minutes. Reaction products were resolved by 15% SDS-PAGE and immunoblotted with antiphospho GSK-3 antibody.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/96/3/10.1182_blood.v96.3.941/5/bloo01514003w.jpeg?Expires=1726829943&Signature=b5tsv~DjbyeNvgtfQr-dkiFeyqdPVL-qL7--b7U-Euoh0msu1786QPvE60OgefpRw6tbJCadhuxdujo-fvOVBYa9ns45pj06pkKdtE9i8ZhC1i6pmZiERvmzJRL1g1x5V4tisuB3hYDy1yHFeya4clMJU8N4XnHgozi8YVEmwrhBm~oZULnhQX6T8sxEIwfS9uzjrnfWQrPH4wjdXIz61IV68NRxaUsRdblrJWJSXqsV1kkuGQvBczOrBfaAPMDvn~xjia4beDOhPKqpJzbfWlYxGaemBgm19X1UDwKgC59tdet6MSjqLGKmhyhHE2jKXJ35CCajymNnLwJiG-xpNQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
In vitro kinase assay revealed that AKT kinase is activated by EPO stimulation.
(A) EPO was removed from UT-7/EPO cells for 24 hours. Then the cells were stimulated with EPO (10 U/mL) for 10 minutes. After solubilization, cell lysates were immunoprecipitated with protein G-conjugated anti-Akt antibody. Then immunoprecipitates were subjected to an in vitro kinase assay. The kinase assays were carried out in the presence of 10 μCi of [γ-32P]ATP (3000 Ci/mmol) using histone H2B as a substrate (0.1 mg/mL). The reactions were incubated at 25°C for 30 minutes. Reaction products were resolved by 15% SDS-PAGE and visualized by autoradiography. (B) EPO was removed from UT-7/EPO cells for 24 hours, then the cells were incubated with 50 μmol/L LY294002 for 45 minutes and stimulated with EPO (10 U/mL) for 10 minutes. Cells were lysed and immunoprecipitated with anti-Akt antibody. Then immunoprecipitates were subjected to an in vitro kinase assay. The kinase assays were carried out in the presence of 200 μmol/L ATP using GSK-3 as a substrate (0.025 mg/mL) . The reactions were incubated at 30°C for 30 minutes. Reaction products were resolved by 15% SDS-PAGE and immunoblotted with antiphospho GSK-3 antibody.
