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. Author manuscript; available in PMC: 2019 Sep 1.
Published in final edited form as: Cancer Immunol Res. 2018 Jul 6;6(9):1008–1013. doi: 10.1158/2326-6066.CIR-18-0102

Figure 2. Discovery of Siglec-6 as the antigen of mAb JML-1.

Figure 2

A, the binding of biotinylated JML-1 IgG1 (red) and a biotinylated human IgG1 isotype control (light blue) to MEC1 (left), MEC1-002 (middle), and U937 (right) cell lines was detected with PE-conjugated streptavidin by flow cytometry. A representative out of three independent experiments is shown. B, listed are the top ten RNA-seq hits of genes encoding human transmembrane proteins with differential expression in MEC-1 and MEC1-002 cells. Of these, nine were present in the Retrogenix cell microarray of a collection of 4,336 human transmembrane proteins and one (Siglec-6 encoded by SIGLEC6) was detected with JML-1 IgG1 in primary and confirmatory screens. C, MEC1-002 cells were incubated with siRNA targeting SIGLEC6 (red) or a control siRNA (blue) and then analyzed by flow cytometry for binding by either biotinylated JML-1 IgG1 in conjunction with PE-conjugated streptavidin or mouse anti-human Siglec-6 mAb 767329 in conjunction with Alexa Fluor 647-conjugated goat anti-mouse IgG pAbs. Three independent experiments that were carried out are shown as mean ± SD values and analyzed for significance by two-way ANOVA (****, p < 0.0001).