Negative control blots (Fig. 5B and Fig. 6, right panel) generated by substituting mouse IgG1K isotype for the primary antibody were virtually devoid of any bands. As a further control in Western blotting experiments, the 7.9.2 monoclonal antibody was also preabsorbed with soluble EPO-R (Fig. 7). The intensity of the 66-kDa band in TF-1 cells and purified trophoblast cells was considerably reduced compared to that obtained after incubation with 7.9.2 antibody alone, and the EPO-R band was eliminated altogether in placental homogenates. Band intensity was not affected when the EPO-R antibody was preabsorbed with a nonspecific antigen (rhEPO, data not shown).
A larger molecular size band was also apparent (~90 kDa), but it was not diminished by preabsorption of the 7.9.2 monoclonal antibody with soluble EPO-R. In contrast, a lower molecular size band (~45 kDa, possibly the truncated EPO-R; see Discussion) was eliminated by the preabsorption protocol (data not shown). Finally, to ensure that the EPO-R monoclonal antibody was not cross-reacting with cytokeratins, simultaneous detection of EPO-R and placental cytokeratin with a pan-antibody was performed (Fig. 8, A and B). Immunoreactive EPO-R and cytokeratins bands were not overlapping. Although the pan-cytokeratin antibody does not detect cytokeratin 9, which is 64 kDa, this cytokeratin has been found exclusively in the suprabasal epidermis of the footpad.
FIG. 5. Detection of EPO-R on immunoblots of human placental lysates and immunopurified trophoblast cell homogenates using mouse monoclonal antibody mh2er 7.9.2. Proteins were first separated by SDS-PAGE (see Materials and Methods for details). A) Lane 1: isolated, immunopurified first-trimester trophoblast cells; lane 2: isolated, immunopurified third-trimester trophoblast cells; lane 3: first-trimester villous placenta; lane 4: second-trimester villous placenta; lane 5: term villous placenta; lane 6: villous placenta from a woman diagnosed with preeclampsia. B) The negative control blot where the mouse IgG1 к isotype was substituted for the primary antibody (lanes 1-6). Molecular weight standards X 10-3 are indicated on the left.
FIG. 6. Western blot analysis of EPO-R using mouse monoclonal antibody clone mh2er 7.9.2. A prominent 66-kDa band was detected in positive control cell lysates from TF-1 cells (lane 1) and in the trophoblast-derived choriocarcinoma Jar cell lysates (lane 2). No reactivity was detected in negative control HeLa cells (lane 3) or in a negative control blot where the mouse IgG1 к isotype was substituted for the primary antibody (lanes 4-6). Molecular weight standards X 10~3 are indicated on the left.
FIG. 7. Detection of EPO-R in TF-1 cells (lane 1), trophoblast cells isolated and immunopurified from first-trimester placenta (lane 2), and third-trimester villous placental homogenates (lane 3). Immunoblots were detected using either anti-EPO-R monoclonal antibody 7.9.2 (A) or antibody preabsorbed with soluble EPO-R (B). C) The negative control blot where mouse IgG1 к was substituted for the primary antibody. Molecular weight standard X 10-3 is indicated on the left.