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Blood, Vol. 110, Issue 2, 509-518, July 15, 2007
Erythropoietin modulation of podocalyxin and a proposed erythroblast niche
Blood Sathyanarayana et al.
110: 509
Supplemental materials for Sathyanarayana et al, Volume 110, Issue 2, 509-518
Files in this Data Supplement:
- Document 1. Abbreviations list (PDF, 19.5 KB) -
Due to the necessary use of a substantial number of abbreviations, an overall list is provided for this article.
- Figure S1. Additional Epo-modulated genes in murine bone marrow derived erythroblasts (JPG, 121 KB)
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This figure is provided to underline the robust nature of transcriptome analyses, and to illustrate additional specific examples of Epo-modulated genes. A) Epo-regulated genes related to Akt, Erk, and ubiquitin pathways. B) For Epo-regulated ubiquitin related factors, Affymetrix 430-2.0 array--based analyses are graphed. Values are mean fold-modulation by Epo (+/− SE, n=4). Ci) Quantitative RT-PCR analyses of Epo regulation of Btrc, Usp12, Usp36, Siah2, and Usp18 in KitposCD71highTer119neg wt-EpoR erythroblasts. Values are means (+/− SE) and are normalized to beta-actin. Note the approximate four-fold down-modulation of Btrc, a candidate ubiquitin ligase for the EpoR 71. Cii) Quantitative RT-PCR analyses of Epo regulation of Btrc, Usp12, Usp36, Siah2, and Usp18 in KitposCD71highTer119neg EpoR-H erythroblasts. Values are means (+/− SE) and are normalized to beta-actin. Ciii) Quantitative RT-PCR analyses of Epo regulation of Btrc, Usp12, Usp36, Siah2, and Usp18 in KitposCD71highTer119neg EpoR-HM erythroblasts. Values are means (+/− SE) and are normalized to beta-actin.
- Figure S2 (JPG, 64.5 KB)
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These micrographs correspond to Figure 2C, and are provided as larger, more readily inspected, images of Wright stained cytospin preparations of Kit+CD71highTer119−, Kit−CD71highTer119−, and Kit−CD71highTer119+ erythroblasts.
- Figure S3. Cell surface levels of EpoR expression in purified Kit+CD71highTer119− and Kit−CD71highTer119+ erythroblasts (JPG, 47.6 KB)
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Cell surface EpoRs were retrieved from these cohorts of developmentally staged erythroblasts using biotin-sialyl-Epo and streptavidin agarose CL4B. As a control, 30-fold excess of unlabeled Epo was included (+/− for each sample). Retrieved EpoR complexes were then analyzed by western blotting.
- Figure S4. Flow cytometric analyses of reticulocyte maturity, and PODXL positivity, within bone marrow preparations (at day 0.5 after Epo-dosing) (JPG, 94 KB)
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Here, sample data are provided to illustrate the approach used to determine the enucleated fraction of bone marrow reticulocyte subpopulations. Initial gating was on a high forward-angle and side-scatter defined population (log scale, upper left panel). Via DRAQ5 staining, the enucleated DRAQ5− fraction then was identified (blue) within a forward scatter-side scatter plot (upper right panel). A gate was drawn around this enucleated population, and TO positivity then was determined (center panel). Subsequently, PODXL staining (Alexa-647) was determined for each compartment of TO stained enucleated populations (RBCs, mature reticulocytes, IRFs) (lower panels).
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