Figure 4

Mesothelin recognizes oligosaccharides expressed on MUC16. (A) Meso-Fc binding to OVCAR-3-derived MUC16 (200 U CA125/lane) after oxidation with 1 mM (lane 2) or 10 mM (lane 4) SMP was detected in overlay experiments. Matching controls are in lanes 1 and 3, respectively. The blots were sequentially overlaid with meso-Fc, MN and a horseradish peroxidase conjugated secondary. (B) Meso-Fc binding to desailylated (lane 2) and non-desialylated (lane 1) MUC16 (150 U CA125/lane) from OVCAR-3 cells was determined in overlay experiments. (C) Meso-Fc binding to MUC16 (200 U CA125/lane) treated with PNGaseF (lane 2) or with buffer only (lane 1) was determined by Western blot overlay experiments. (D) Inhibition of bacterial mesothelin binding to OVCAR-3-derived MUC16 (200 U of CA125/blot) by ConA (blot 2), WGA (blot 3), and E-PHA (blot 4) detected in overlay experiments (D). No lectin was added in blot 1. (E) MUC16 from patient #2 (lane 1; 100 U of CA125) or from OVCAR-3 (lane 2; 500 U of CA125) were overlaid with meso-Fc in the presence (blot on left) or the absence (blot on right) of 0.5 M α-methylmannopyranoside. MN was used to detect meso-Fc binding in all experiments. Since full gel profiles have been shown in Fig. 1, only partial blots are shown here.