Erythropoietin receptors on cancer cells: exciting perspectives, difficult to appreciate.

In this issue, Verdier et al comment on our recent publication, concluding that some anti-EpoR antibodies have limited utility for detecting EpoR expression. 1 We reported that the C-20 anti-EpoR antibody from Santa Cruz Biotechnology (Santa Cruz, CA) detected 5 protein bands in UT-7/Epo cells including 59-kDa and 66-kDa proteins. Using both direct and indirect methods, we showed that the 59-kDa protein, not the 66-kDa protein, is EpoR: (1) the 59-kDa protein migrated similarly to recombinant, FLAG-tagged, full-length EpoR; (2) the 59-kDa protein levels decreased following EPOR shRNA treatment of cells, whereas 66-kDa protein levels remained unchanged; (3) the 59-kDa protein was absent in EPOR-knockout fetal liver but present in wild-type fetal liver; (4) C-20 immunoprecipitated the 59-kDa protein, not the 66-kDa protein, from UT-7/Epo cells (the immunocomplexed 59-kDa protein was detected by 2 other anti-EpoR antibodies, 07-311 and M-20); (5) the 59-kDa, not the 66-kDa, protein bands contained EpoR peptide sequences; and (6) peptides derived from heat-shock proteins specifically inhibited C-20 binding to the 66-kDa protein. Agreeing that many antibodies are unsuitable for detecting EpoR, the authors also do not challenge our conclusion that C-20 should not be used for immunohistochemistry. However, they disagree about the utility of C-20 for detecting EpoR proteins in Western blots and that the apparent molecular mass of EpoR is approximately 59 kDa, not 66/78 kDa as described in the product information sheet provided by Santa Cruz. Verdier et al claimed that 3 protein bands larger than 59 kDa (64, 67.6, and 69.5 kDa) detected in UT-7 cells by C-20 were different forms of EpoR, based on indirect methods. First, they showed that the 64-kDa protein was not detected by C-20 in EpoR-negative Mo7E cells, although an approximately 68-kDa protein was detected in the same cells. Second, they showed that the levels of these proteins were altered following treatment with cycloheximide or stimulation with Epo. However, neither agent necessarily selectively alters EpoR levels. Third, they used an in-house anti-EpoR antibody (C-236), biotinylated Epo/streptavi-din, and an anti-Epo antibody to immunoprecipitate the putative EpoR proteins, but each yielded different protein patterns. For example, 2 proteins were immunoprecipitated by C-236 and only one by biotinylated Epo/streptavidin or the anti-Epo antibody. Furthermore, when probed with a second batch of C-20 (lot B2105), the negative control anti-GST antibody appeared to precipitate the same 2 putative EpoR proteins as C-236. Finally, they showed a predominant 64-kDa protein in BaF3 cells …