sEpoR is regulated by proinflammatory cytokines.

<p><b>4a.</b> IL-6, TNF-α and PMA increase sEpoR in the supernatant of K562 cells. K562 were plated in serum free media and exposed for 48 h to vehicle, PMA, IL-6 and TNF-α. At the end of the incubation cells were pelleted and the supernatant subjected to ELISA for sEpoR. sEpoR measurements were corrected for total protein concentration. * represents p value of <0.05 when compared to the control group. <b>4b.</b> Mean IL-6 levels in subjects with low (n = 32) and high sEpoR (n = 32) are shown. * represents p value of <0.05 when compared to low sEpoR group.</p

Functional characterization of sEpoR.

<p><b>3a.</b> Western blot showing increasedphospho-Stat-5 in the presence of increasing erythropoietin (25 to 5000 mU/ml) in BaF3/EpoR cell lysates. <b>3b</b>. Representative western blot showing total phospho-Stat-5 and Stat-5 in the presence of erythropoietin 5000 mU/ml and varying concentrations of recombinant sEpoR-Fc (50 -5000 ng/ml). Phospho-Stat-5 decreases with increasing recombinant sEpoR. <b>3c</b>. Quantification of sEpoR-Fc inhibition of phospho-Stat 5 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009246#pone-0009246-g003" target="_blank">Figure 3b</a>). Ratios of phospho/total Stat-5 (mean ± SD, n = 3) represented as a percentage of control (Epo alone). *represents p<0.05. <b>3d</b>. Serum from patients with high sEpoR blocks erythropoietin mediated Stat-5 phosphorylation. Shown is the ratio of phospho-Stat-5 to Stat-5 as measured by densitometry. Serum starved BaF3/EpoR cells were exposed to vehicle (negative control), erythropoietin at 50 mU/ml (positive control) and erythropoietin plus 10% serum with Low sEpoR (≤62.5 pg/ml) or serum with high sEpoR (≥4000 pg/ml) for 10 minutes. Cells were lysed in RIPA buffer and 10 ug protein/lane was run on a 4-12% denaturing gel. Gels were transferred and blotted with anti-Stat-5 and anti-phospho-Stat 5. <b>3e</b>. Quantification of western data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009246#pone-0009246-g003" target="_blank">Figure 3d</a>). Ratios of phospho/total Stat-5 (mean ± SD, n = 5 individual patient samples each for low sEpoR and high sEpoR) represented as a percentage of control (Epo alone). *represents p<0.05.</p

sEpoR characterization in uremic serum.

<p><b>1a</b>. Soluble EpoR is detectable in serum from dialysis patients by western blot. Human serum was subjected to immunoprecipitation with goat anti-human erythropoietin receptor antibody (R&D Systems, AF-322-PB) followed by western blotting with mouse monoclonal anti-human erythropoietin receptor (R&D Systems, MAB307). Both antibodies recognize the extracellular domain of the receptor. Lanes 1–6 are serum from 6 representative dialysis patients, lane 7 is blank and lane 8 is recombinant sEpoR (Sigma Aldrich E0643, Saint Louis MI). Shown in the serum samples is a band of expected molecular weight of approximately 27 kDa. The control sEpoR with Fc tag is consistent with the manufacturers reported molecular weight of 32 kDa. <b>1b</b>. Soluble EpoR is also detected using the same dialysis patient serum samples by performing immunoprecipitation in reverse order. In this experiment immunoprecipitation was done with mouse monoclonal anti-human erythropoietin receptor (R&D Systems, MAB307) followed by western blotting with goat anti-human erythropoietin receptor (R&D Systems, AF-322-PB). Lanes 1 to 3 are serum from 3 dialysis patients, and lane 4 is recombinant sEpoR-Fc (Sigma, 307) as positive control.</p