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Figure 19 (A) Schematic representation of a murine dihydrofolate reductase (DHFR) protein complementation assay. Erythropoietin receptor (Epo R) extracellular and transmembrane domains [EpoR(1-270)] are fused to each of the two complementary fragments of DHFR, F[1,2] or F[3], and stably cotransfected in CHO cells. Contransfectants grown in the presence of 2 nM Epo in nucleotide-free medium are selected and incubated with DHFR inhibitor fluorescein-conjugated methotrexate (fMTX). In the presence of Epo or peptide agonist EMP1, dimerization of EpoR(1-270)-F[1,2] and EpoR(1-270)-F[3] reconstitutes DHFR to which fMTX is bound and can be detected in FACS or fluorescence spectroscopy. (B) Schematic representation of P-galactosidase protein complementation assay in intact eukaryotic cells. (1) When the P-gal mutants Aa and Aro are fused to proteins that do not dimerize, the association to active P-gal is not favored. (2) When the Aa and Aro mutants are fused to proteins-that can dimerize, the formation of active P-gal results. P-Gal activity can be measured by chemiluminescence.

Figure 19 (A) Schematic representation of a murine dihydrofolate reductase (DHFR) protein complementation assay. Erythropoietin receptor (Epo R) extracellular and transmembrane domains [EpoR(1-270)] are fused to each of the two complementary fragments of DHFR, F[1,2] or F[3], and stably cotransfected in CHO cells. Contransfectants grown in the presence of 2 nM Epo in nucleotide-free medium are selected and incubated with DHFR inhibitor fluorescein-conjugated methotrexate (fMTX). In the presence of Epo or peptide agonist EMP1, dimerization of EpoR(1-270)-F[1,2] and EpoR(1-270)-F[3] reconstitutes DHFR to which fMTX is bound and can be detected in FACS or fluorescence spectroscopy. (B) Schematic representation of P-galactosidase protein complementation assay in intact eukaryotic cells. (1) When the P-gal mutants Aa and Aro are fused to proteins that do not dimerize, the association to active P-gal is not favored. (2) When the Aa and Aro mutants are fused to proteins-that can dimerize, the formation of active P-gal results. P-Gal activity can be measured by chemiluminescence.

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