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. 2020 Sep 12;21(18):6672. doi: 10.3390/ijms21186672

Figure 4.

Figure 4

Functional characterization of S2-expressed 5D3 variants. (A) Thermal stability was determined using nanoDSF. Melting temperature curves and corresponding numerical values are shown. (B) Specificity of 5D3 variants evaluated by indirect immunofluorescence microscopy. PC-3 PIP (PSMA-positive) and parent PC-3 (PSMA-negative) cells were fixed by formaldehyde and permeabilized by Triton X-100 prior to incubation with recombinant 5D3 variants and secondary antibodies (green channel). Cell nuclei were counterstained with DAPI (blue channel). The scale bar represents 25 µm. (C) Flow cytometry analysis of PSMA-positive (PC-3 PIP, LNCaP) and negative (PC-3, DU145) cells stained by 100 nM purified 5D3 variants. Each gated population represents approximately 30,000 viable cells. (D,E) Determination of binding affinity of 5D3 variants by native ELISA (D) and flow cytometry (E) using purified PSMA and live LNCaP cells, respectively. PBS and DU145 cells were used as negative controls for ELISA and flow cytometry samples, respectively, and corresponding background signals were subtracted.