Year: 2006
Type: letter
Abstract: Anti-Epo receptor antibodies do not predict Epo receptor expressionIn 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, FLAGtagged, 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
Source: Blood
Authors: Michael Henke, Ajay Verma, Geza Acs
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W1976699756