BFU-E colonies express EPO protein. Bone marrow mononuclear cells were plated in semisolid media with either EPO or EMP. (A) BFU-E and CFU-GM colonies were pooled, washed, and lysed with Triton X-100–based lysis buffer. Lysates (20 μg per lane) were separated on 10% SDS-PAGE, transferred onto nitrocellulose membrane, and incubated with rabbit polyclonal anti-EPO antibody. A single band with comparable electrophoretic mobility to rhEPO (lane 1) was detected in BFU-E cells generated with EMP1 (lane 2) or with rhEPO (lane 3). The EPO-specific band was absent in Epstein-Barr virus–transformed lymphocytes (lane 4), granulocytes (lane 5), monocytes (lane 6), and CFU-GM colonies (lane 7). The same membrane was reprobed with antibodies against abundantly expressed angiotensin receptor type 1 (also expressed in BFU-E colony cells)26and a specific band of comparable intensity was detected in all samples. (B) Coomassie staining of protein lysates used on Western blots. To see electrophoretic migration rhEPO was loaded in excess of 50 U/lane.