Figure 5
Figure 5. Glycan structures on PSGL-1. (A) HL-60 cells were cultured in the presence of 50μM acetylated monosaccharides (4F-GalNAc, 4F-GlcNAc, GalNAc) for 36 hours as outlined in Figure 2A through H. PSGL-1 was immunoprecipitated from 400 μg of cell lysate onto TB5 beads. Carbohydrate epitopes on this antigen were then probed using HECA-452 and DSA lectin. The number of immobilized PSGL-1 was quantified using anti-PSGL-1 polyclonal mAb H-300. Black-empty and color-filled histograms represent the fluorescence of beads resulting from Ab/lectin in the absence and presence of cell lysate, respectively. Dashed vertical line indicates peak fluorescence intensity in vehicle control. Representative histograms are shown. (B) Summary of results from panel A for 3 or more independent runs. Normalized geometric mean fluorescence intensity (%) = (MFI of sample − background MFI in the absence of cells)/(MFI of vehicle control − background MFI in the absence of cells). 4F-GalNAc reduces CLA/HECA expression on PSGL-1 and the amount of DSA-lectin recognition. *P < .05 with respect to GalNAc treatment. (C-D) Representative cytometry-bead histograms (C) and normalized MFI data (D) show temporal changes in PSGL-1 glycosylation on 50μM 4F-GalNAc or GalNAc treatment. Here, HL-60 cells were cultured in the absence of prior neuraminidase treatment (protocol in Figure 2I-L). CLA/HECA-452 expression, DSA-lectin binding, and PSGL-1 amount on beads were quantified after capture of PSGL-1 from same amount of lysate in all samples. *P < .05 with respect to t = 0 hours. (E) PSGL-1 from HL-60 cells cultured in 50μM 4F-GalNAc, 4F-GlcNAc, or GalNAc were immunoprecipitated using Ab H-300 and analyzed using Western blot analysis. Protein immunoprecipitated from 300 μg of lysate was loaded in each lane of 2 identical 8% polyacrylamide gel electrophoresis gels. One blot was probed with anti-PSGL-1 monoclonal antibody (KPL-1) and the other with HECA-452. CLA/HECA expression on PSGL-1 was reduced on 4F-GalNAc treatment. (F) 4F-GalNAc treatment reduced the molecular weight of PSGL-1 by approximately 10% to 15%.

Glycan structures on PSGL-1. (A) HL-60 cells were cultured in the presence of 50μM acetylated monosaccharides (4F-GalNAc, 4F-GlcNAc, GalNAc) for 36 hours as outlined in Figure 2A through H. PSGL-1 was immunoprecipitated from 400 μg of cell lysate onto TB5 beads. Carbohydrate epitopes on this antigen were then probed using HECA-452 and DSA lectin. The number of immobilized PSGL-1 was quantified using anti-PSGL-1 polyclonal mAb H-300. Black-empty and color-filled histograms represent the fluorescence of beads resulting from Ab/lectin in the absence and presence of cell lysate, respectively. Dashed vertical line indicates peak fluorescence intensity in vehicle control. Representative histograms are shown. (B) Summary of results from panel A for 3 or more independent runs. Normalized geometric mean fluorescence intensity (%) = (MFI of sample − background MFI in the absence of cells)/(MFI of vehicle control − background MFI in the absence of cells). 4F-GalNAc reduces CLA/HECA expression on PSGL-1 and the amount of DSA-lectin recognition. *P < .05 with respect to GalNAc treatment. (C-D) Representative cytometry-bead histograms (C) and normalized MFI data (D) show temporal changes in PSGL-1 glycosylation on 50μM 4F-GalNAc or GalNAc treatment. Here, HL-60 cells were cultured in the absence of prior neuraminidase treatment (protocol in Figure 2I-L). CLA/HECA-452 expression, DSA-lectin binding, and PSGL-1 amount on beads were quantified after capture of PSGL-1 from same amount of lysate in all samples. *P < .05 with respect to t = 0 hours. (E) PSGL-1 from HL-60 cells cultured in 50μM 4F-GalNAc, 4F-GlcNAc, or GalNAc were immunoprecipitated using Ab H-300 and analyzed using Western blot analysis. Protein immunoprecipitated from 300 μg of lysate was loaded in each lane of 2 identical 8% polyacrylamide gel electrophoresis gels. One blot was probed with anti-PSGL-1 monoclonal antibody (KPL-1) and the other with HECA-452. CLA/HECA expression on PSGL-1 was reduced on 4F-GalNAc treatment. (F) 4F-GalNAc treatment reduced the molecular weight of PSGL-1 by approximately 10% to 15%.

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