American Society of Hematology

Figure 3.

$Figure 3. GpC restricts Lu/BCAM activity. (A) BRIC4, 10, and 100 were used to immunoprecipitate (IP) GpC from an erythrocyte lysate. Only BRIC10 was able to co-IP Lu/BCAM, suggesting that Lu/BCAM may mask the sialylated GpC epitope recognized by BRIC4 and possibly BRIC100 as well. Anti-CD235 was used to IP glycophorin-A (GpA). Western blotting for Lu/BCAM shows that BRIC10 co-IPs Lu/BCAM (top arrow). The anti-goat horseradish peroxidase–linked antibody directed against primary Lu/BCAM antibody was found to cross-react with the mouse light chain of the BRICS used to IP GpC (lower arrow). (B) Confocal micrograph showing Lu/BCAM clusters (fluorescein isothiocyanate, green; original magnification ×40) partially colocalizing (yellow, indicated with white arrowheads) with GpC (BRIC10, PE, red). (C) Western blot of GpC immunoprecipitation from Lu-WT and Lu-R338A transfected cells. GpC was found to co-IP only with Lu-WT. (D) GpC-ex3 (Gerbich phenotype GPC) sialylation was quantified by flow cytometry using BRIC4 and is expressed as a percentage compared with control erythrocytes (x-axis). GpC sialylation was then plotted against adhesion frequency to laminin-α5 (n = 5), which was also normalized to control (y-axis). The squared correlation coefficient (R2) is indicated. (E) Gerbich phenotype erythrocyte adhesion negatively correlates with Lu/BCAM expression. (F) Flow cytometric comparison, using BRIC4 and goat anti-human Lu/BCAM (R&D Systems), of Lu−GpC− (orange), Lu+GpC− (red), and Lu+GpC+ (blue) transfected HEK293T cells. (G) Adhesion frequency of Lu+GpC− (normalized to 1) and Lu+GpC+ HEK293T cells to laminin-α5. (H) Of the HEK293T transfected with both Lu and GpC (blue population), 5.2% failed to express GpC but did express Lu (blue population in red gate, right lower quadrant), as was assessed by flow cytometry. Upon flowing the total HEK293T cell population over laminin-coated ibidi chambers, the 5.2% GpC negative fraction was significantly enriched for on laminin-α5–coated ibidi chambers as determined by fluorescence microscopy (n = 4). *P < .05.$

GpC restricts Lu/BCAM activity. (A) BRIC4, 10, and 100 were used to immunoprecipitate (IP) GpC from an erythrocyte lysate. Only BRIC10 was able to co-IP Lu/BCAM, suggesting that Lu/BCAM may mask the sialylated GpC epitope recognized by BRIC4 and possibly BRIC100 as well. Anti-CD235 was used to IP glycophorin-A (GpA). Western blotting for Lu/BCAM shows that BRIC10 co-IPs Lu/BCAM (top arrow). The anti-goat horseradish peroxidase–linked antibody directed against primary Lu/BCAM antibody was found to cross-react with the mouse light chain of the BRICS used to IP GpC (lower arrow). (B) Confocal micrograph showing Lu/BCAM clusters (fluorescein isothiocyanate, green; original magnification ×40) partially colocalizing (yellow, indicated with white arrowheads) with GpC (BRIC10, PE, red). (C) Western blot of GpC immunoprecipitation from Lu-WT and Lu-R338A transfected cells. GpC was found to co-IP only with Lu-WT. (D) GpC-ex3 (Gerbich phenotype GPC) sialylation was quantified by flow cytometry using BRIC4 and is expressed as a percentage compared with control erythrocytes (x-axis). GpC sialylation was then plotted against adhesion frequency to laminin-α5 (n = 5), which was also normalized to control (y-axis). The squared correlation coefficient (R²) is indicated. (E) Gerbich phenotype erythrocyte adhesion negatively correlates with Lu/BCAM expression. (F) Flow cytometric comparison, using BRIC4 and goat anti-human Lu/BCAM (R&D Systems), of Lu⁻GpC⁻ (orange), Lu⁺GpC⁻ (red), and Lu⁺GpC⁺ (blue) transfected HEK293T cells. (G) Adhesion frequency of Lu⁺GpC⁻ (normalized to 1) and Lu⁺GpC⁺ HEK293T cells to laminin-α5. (H) Of the HEK293T transfected with both Lu and GpC (blue population), 5.2% failed to express GpC but did express Lu (blue population in red gate, right lower quadrant), as was assessed by flow cytometry. Upon flowing the total HEK293T cell population over laminin-coated ibidi chambers, the 5.2% GpC negative fraction was significantly enriched for on laminin-α5–coated ibidi chambers as determined by fluorescence microscopy (n = 4). *P < .05.

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