In addition, we report for the first time Epo induced Ras activation in PEPs. Surprisingly, Ras activation is also dependent on PI3K activity. This differs tremendously in the signaling mechanism for Ras in lots of other contexts, exactly where class Ia PI3Ks are downstream of Ras. The new pathway described here could possibly be independent of phos photyrosyl SH2 domain interactions which are seen in quite a few receptor signaling pathways. This would explain why mice having a mutant Epo receptor that lacks all SH2 domain docking web sites are nevertheless viable and possess a normal hematocrit when unchallenged. Approaches Purification and amplification of primary human erythroid progenitors Key human erythroid progenitors have been purified as described in Freyssinier et al. Briefly, umbilical cord bloods were diluted 1,1 with phosphate buffered saline containing 4% fetal bovine serum.
Right after Histopaque 1077 density gradient centrifugation, the low den sity cells had been recovered and CD34 cells purified by immunomagnetic cell separation. Selected CD34 cells have been cultured in 5% CO2 at 37 C for seven days in serum no cost RM B00 cell expan sion medium with 10 ng ml interleukin 3, a fantastic read ten ng ml IL six and 25 ng ml SCF. Soon after seven days in culture, CD36 cells have been immu nomagnetically isolated using a mouse anti CD36 mAb and rat anti mouse IgG1 microbeads. CD36 cells were then further cultured with IL three, IL six, SCF and two U ml Epo for 4 days. Cell stimulation, lysis and protein quantification On day 7 four CD36 cells were starved for 6 h in RM B00 medium and stimulated with 0. three U ml Epo, 25 ng ml SCF or 100 M TPA for 10 min, unless indicated otherwise.
Mock incubations had been accomplished together with the acceptable solvents for cytokines or chem icals. For lipid and protein kinase inhibition experiments, cells have been pretreated with UO126, wortmannin, caffeine, LY294002, ZM336372, calphostin C or Ro 31 8220 as indicated. Total cell protein extracts were prepared by solubilizing in Igepal CA 630 lysis buffer containing 1% NP 40, 50 mM HEPES pH NXY059 7. 5, 10% glycerol, 150 mM NaCl, 2 mM EDTA, 50 mM sodium fluoride, ten mM sodium pyruvate, 1 mM sodium vanadate, 1 mM phenylmethylsulfo nylfluoride, 10g ml aprotinin, 5g ml antipain, 0. 5g ml leupeptin, 0. 7g ml pepstatin A, 1 Full inhibitors and 3 mM dithiotreitol. Soon after 30 min of nutation inside the cold, lysates were cleared by centrifugation for 20 min at two C with 20,000 g and supernatants fur ther analyzed.
Protein concentrations had been determined together with the Bradford dye binding assay for all extracts produced. Immunoblotting and immunoprecipitation 100g of total cell protein extract had been separated by SDS polyacrylamide gel electrophoresis. Just after membrane transfer proteins of interest were detected with antibodies against phospho STAT5a b, phosphotyrosine, Ras, phospho Erk1 2, phospho MEK1 two, phospho Akt, phospho GSK3 , Akt or Erk1 2 and ECL.