Selected anti-Epo receptor antibodies predict Epo receptor expression.

In an article recently published in Blood, Elliott et al 1 reported that commercially available anti–erythropoietin receptor (anti-EpoR) antibodies are hardly usable to predict EpoR expression because of their low specificity and affinity. Although we agree with the authors that many of these antibodies work poorly, we strongly disagree with them concerning 2 main points: (1) the ability of Santa Cruz Biotechnology C-20 antibody (Santa Cruz, CA) to efficiently detect the EpoR in Western blot (WB) analysis, and (2) the apparent molecular mass of the EpoR. For a long time, we have been using C-20 anti-EpoR antibodies from Santa Cruz Biotechnology (catalog no. SC-695) to reveal EpoR in WB (see, for example, Verdier et al 2 and Walrafen et al 3), and we always detect EpoR with a high sensitivity and a good specificity. To demonstrate the efficiency of this antibody, we used UT-7 cells, since they express endogenous EpoR and they were used by Elliott et al. 1 The Mo7E cell line was used as a negative control. In UT-7 cells, C-20 antibodies revealed 5 bands with molecular masses of 110 (a), 69.5 (b), 67.6 (c), 64 (d), and 58 kDa (e). In Mo7E cells, only bands a and e were clearly visible, with a faint band at 68.4 kDa (f) that was masked by bands b and c in UT-7 cells (Figure 1). We identified bands b, c, and d as the EpoR because they are not present in Mo7E extracts (Figure 1A-B, lane 2); because as the EpoR 2 they are short-lived proteins (Figure 1A, lane 5); and because they are immunoprecipitated by another anti-EpoR antibody (C-236 3 ; Figure 1A, lane 7). Bands b and c represent the cell surface form of the EpoR, since they are shifted after Epo stimulation (Figure 1A-B lanes 1 and 3), they are downregulated by sustained Epo stimulation (Figure 1A, lane 4), and they are precipitated by biotinylated Epo/streptavidin (Figure 1A, lane 6) and by Epo/anti-Epo antibodies (Figure 1A, lane 9). Band d, which is strongly increased in EpoR-transfected BaF3 cells, corresponds to the maturing EpoR present in the endoplasmic reticulum 3 (Figure 1C). To explain the difference between the results published by Elliott et al 1 and ours, we suspected that the antibody batches were different. We tested the same extracts using another batch (B2105). This latter batch revealed mainly bands a, e, and f, which are contaminating proteins …