Figure 3 | Scientific Reports

Figure 3

From: Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay

Figure 3

Epitope mapping using swapping DRD1 mutants.

(a) A schematic diagram of epitope mapping using swapped mutant. An arbitrary part of DRD1 was swapped with homologous region of other GPCRs by PCR and In-Fusion assembly. Binding between mAb clones and swapped mutants was evaluated by BiLIA. (b) Binding of mAbs toward swapped mutants. The left of figure shows swapped region (red area) and transmembrane (shaded box) of each swapped clone. Intense blue in the right plots indicates that the mAb did not bind to the swapped mutant. (c) Staining profiles of DRD1-overexpressed cell with DRD1-specific antibodies. HeLa cells were transfected with pIRES2-AcGFP/DRD1 and stained with DRD1-specific antibodies 3C9 (left) or Ra51 (right) followed by Alexa Fluor 647-conjugated anti-mouse (left) or anti-rabbit (right) IgG-specific antibody, respectively. Stained cells were analyzed with a flow cytometer. Transfected cells were gated on the basis of GFP expression. Horizontal axis indicates the log intensities of fluorescence; vertical axis indicates cell number. Representative histograms in two independent experiments are shown.

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