Expression of the Erythropoietin Receptor: RESULTS(3)


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.
Fig5Expression of the Erythropoietin
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.

Fig6Expression of the Erythropoietin
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.

Fig7Expression of the Erythropoietin
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.

This entry was posted in Human Placenta and tagged Erythropoietin Receptor, Human Placenta, Trophoblast Cells.