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Wu, J. et al.

Assessment of recombinant porcine follicle-stimulating hormone receptor using a novel polyclonal ectodomain antibody.

Follicle-stimulating hormone (FSH) receptors ( FSHR) are critically involved in mediating the responses of granulosa cells and Sertoli cells to FSH. The dynamic changes in cell surface FSH receptors ( FSHR) in response to FSH remain unclear in part because of the heavy reliance on ligand-binding methodologies. This study was designed to determine the molecular and cellular properties of recombinant porcine FSHR using a novel, high-affinity purified polyclonal antibody to the ectodomain of the pFSHR. A full-length porcine FSHR cDNA was cloned and sequenced and recombinant pFSHR protein was stably expressed in a clonal cell line of Chinese hamster ovary cells (pFSHR-CHO). Recombinant receptor was stably expressed in an ovarian cell line with a density similar to that of porcine ovarian cells. A specific polyclonal antibody was generated in chickens to a 100-amino acid fragment of the pFSHR ectodomain. Immunoblotting, immunoprecipitation, indirect immunofluorescence cytochemistry and immunoelectron microscopy were performed using affinity-purified antibody to identify recombinant pFSHR in pFSHR-CHO cells. Immunoblotting of solubilized pFSHR-CHO proteins and immunoprecipitation of pFSHR-CHO protein metabolically labeled with 35S identified a single 74-kDa band in pFSHR-CHO cells; no bands were visualized in mock-transfected CHO cells. Indirect immunofluorescent labeling revealed the presence of pFSHR in pFSHR-CHO cells but not in mock-transfected CHO cells. Immunoelectron microscopy revealed the highest density of pFSHR associated with the plasma membrane and no pFSHR in mock-transfected CHO cells. The chicken anti-pFSHR antibody is a valuable tool for detecting and monitoring of FSHR using a variety of methodologies.[1]

References

Assessment of recombinant porcine follicle-stimulating hormone receptor using a novel polyclonal ectodomain antibody. Wu, J., Wang, Y., Xiao, W., Meyer, K.B., Schmidt, K.M., Morris, R.E., Degen, S.J., La Barbera, A.R. Endocr. Res. (2004) [Pubmed]

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