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Figure 5 | Molecular Cancer

Figure 5

From: Identification of Siglec-9 as the receptor for MUC16 on human NK cells, B cells, and monocytes

Figure 5

Siglec-9 binds to sialylated glycans of sMUC16. A, Fetuin (lane 1) or sMUC16 (300 U of CA125, lane 2; 200 U of CA125, lane 3; and 100 U of CA125, lane 4) were separated by SDS-PAGE, transferred to a PVDF membrane and overlaid with Siglec-9-human Fc chimera (2.5 nM). Binding of Siglec-9-human-Fc was detected using horseradish-peroxidase-labeled mouse anti-human-Fc secondary antibody. B, sMUC16 (250 U of CA125/lane) purified from the spent media of OVCAR-3 cells was loaded in duplicate on both blots. Siglec-9-human-Fc chimera was overlaid on the membrane in the presence of 7.5 nM asialofetuin (left panel) or fetuin (right panel). C, sMUC16 was desialylated with neuraminidase from Clostridium perfringens. Binding of neuraminidase treated and untreated sMUC16 samples (250 U of CA125/lane) to VK-8 (left panel) or Siglec-9-human-Fc (right panel) was detected by Western blotting. Red arrows in all panels indicate interface between the stacking and separating gels. sMUC16 is typically retained in the stacking gel under the electrophoresis conditions used to develop these western blots. Migration of the 200 kDa (A-C) and 60 kDa (A) molecular weight markers is shown.

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