Human Erythropoietin R Antibody

Detection of Human Erythropoietin R by Western Blot. Western blot shows lysates of UT-7 human acute myeloid leukemia cell line. PVDF membrane was probed with 2 µg/mL of Mouse Anti-Human Erythropoietin R Monoclonal Antibody (Catalog # MAB307) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). For additional reference, PVDF membrane was probed with 1 µg/mL of Goat Anti-Human Erythropoietin R Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-322-PB) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). A specific band was detected for Erythropoietin R at approximately 70 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Erythropoietin R in TF‑1 Human Cell Line by Flow Cytometry. TF-1 human erythroleukemic cell line was stained with Mouse Anti-Human Erythropoietin R Monoclonal Antibody (Catalog # MAB307, filled histogram) or isotype control antibody (MAB0041, open histogram) followed by anti-Mouse IgG Allophycocyanin-conjugated Secondary Antibody (F0101B). Staining was performed using our Staining Membrane-associated Proteins protocol.

Detection of Human Erythropoietin R by Western Blot sEpoR characterization in uremic serum.1a. 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. 1b. 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. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/20169072), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Erythropoietin R by Western Blot The 59 kDa protein detected by M-20 In MCF-7 cells is not bound by other anti-EpoR antibodies.The indicated lysates were immunoprecipitated (IP) then the immunoblotted (IB) with the indicated antibodies: ab10653 (abcam Inc), Mab307 (R&D systems), C-20 & M-20 (Santa Cruz Inc) or A-82 (Amgen Inc). COS cell lysates expressing a FLAG-tagged version of EpoR (FLAG-EpoR) [6] and UT-7/Epo cells served as EpoR positive controls. 769-P cells served as the EpoR negative control. (A) Westerns were immunoprecipitated (IP) with ab10653 or M-20 followed by immunoblotting (IB) with M-20. The position of full-length 59 kDa EpoR in positive controls is indicated by the arrow. Positions of molecular weight markers (kDa) are shown. Bands detected in 769-P lysates are non-EpoR cross-reacting proteins and include antibody chains that were not removed completely or protein G that leached from beads. Note the detection of a 59 kDa band with MCF-7 cells with the M:20/M:20 combination but not with the ab10653/IB:M-20 combination. (B) IP:IB combinations with the indicated antibodies were subjected to western analysis. The western slice containing the 59 kDa EpoR band from each combination is shown. Note the 59 kDa bands detected in EpoR positive controls but not 769-P cells. Only the M-20:M-20 combination detected a 59 kDa band in MCF-7 cells. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0068083), licensed under a CC-BY license. Not internally tested by R&D Systems.