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. 2023 Oct 11;13:17218. doi: 10.1038/s41598-023-44316-y

Figure 1.

Figure 1

Characterization of viral E3/49K and pUL11 after transfection in porcine cells. (A) Expression in porcine L23 cells. L23-E3/49K (left) and L23-UL11 cells (right), shown as representative histograms, were stained with anti-E3/49K or anti-pUL11 monoclonal antibodies (mAbs), respectively (solid lines). Dashed-line histograms were obtained after staining of L23-control cells by the mAbs. The numbers represent mean fluorescence intensity. (B) Binding of CD45-His to transfected cells. Purified soluble CD45-His was used for staining of L23-E3/49K and L23-UL11 cells. Binding of soluble molecules to the cells was detected by incubation with PE-conjugated anti-CD45 mAb. Representative histograms show staining under optimal conditions. The numbers represent mean fluorescence intensity. The bar graph shows concentration dependent binding of CD45-His. The results of 1 of 2 similar experiments are shown. (C) Production of soluble E3/49K (sec49K) by transfected cells. Supernatants were harvested from L23-control cells (dotted line) or L23-E3/49K cells (solid line) at different time points after starting the culture. Human PBMC were incubated with supernatants followed by anti-E3/49K staining. Supernatants were also harvested from L23-E3/49K cells cultured for 6h without (solid line) or with Marimastat (filled histogram). The dashed line represents staining with secondary mAb alone. Representative histograms are shown and the numbers represent mean fluorescence intensity. (D) Binding of E3/49K and pUL11 to human CD45. Purified soluble E3/49K (sec49K-His) or pUL11 (UL11-Fc fusion protein) were used for staining of porcine and human PBMC, the mouse B cell line 300–19, and 300–19 cells transfected with the human CD45-ABC isoform. Binding of soluble molecules to the cells was detected by incubation with anti-E3/49K and anti-pUL11 mAb followed by a second incubation with fluorochrome-labelled secondary reagents. Dashed lines in the representative histograms represent background staining with the secondary antibodies alone.

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