EpoRm has higher and more stable expression than EpoR. (A) Western blot analysis of EpoR expression in 293T cells. Cell lysates of 293T cells transduced with EpoR, EpoRm, or GFP only were separated on a 10% polyacrylamide gel under reducing condition. The blotted membrane was probed with mouse anti-Flag antibody (9A3; Cell Signaling Technology, Danvers, MA) followed by goat anti-mouse immunoglobulin G conjugated to horseradish peroxidase (R&D Systems); rabbit anti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EPR16891; Abcam, Cambridge, UK) was used to detect GAPDH (loading control). Antibody binding was revealed by Clarity Western ECL substrate (Bio-Rad, Hercules, CA) and visualized by ChemiDoc Touch Imager (Bio-Rad). (B) Flow cytometric analysis of T lymphocytes transduced with EpoR, EpoRm, or GFP only. Flow cytometric dot-plots illustrate EpoR expression as detected by a PE-conjugated anti-EpoR antibody (R&D Systems). Percentage of cells in each quadrant is shown. (C) Percentage of GFP⁺ T cells expressing EpoR (left) or MFI of EpoR (right) of T cells transduced with EpoR or EpoRm. ****P < .0001. (D) Percentage of CD4⁺ or CD8⁺ T cells from 6 donors expressing EpoR. ****P < .0001. (E) Relation between MFI of GFP and MFI of EpoR in T cells from 3 donors transduced with either EpoR or EpoRm. ****P < .0001. (F) T lymphocytes from 3 donors were transduced with EpoR or EpoRm and then stimulated with 10 IU/mL Epo. Expression of surface EpoR was assessed by flow cytometry at the indicated time points after stimulation.