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1.
FIGURE 3.

FIGURE 3. From: Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue.

Siglec-F ligands in BAL fluid. A, fractions of BAL fluid assayed by ELISA using Siglec-F-Fc (filled circles), or anti-MUC5B (filled squares). Total protein was determined by measuring absorbance at 280 nm (dotted line). No signal was observed when wells were reacted with CD22-Fc (open squares), human IgG (open triangles), or treated with sialidase before incubation with Siglec-F-Fc (open circles). Results are representative of two independent experiments.

Michael L. Patnode, et al. J Biol Chem. 2013 September 13;288(37):26533-26545.
2.
FIGURE 4.

FIGURE 4. From: Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue.

Siglec-F ligand expression during N. brasiliensis infection. A, cryostat-cut serial sections of lungs from N. brasiliensis infected or uninfected mice. Sections were treated with sialidase or buffer alone, then stained with Siglec-F-Fc (green), anti-CD11b (red), and DAPI (blue). B, high power images of the same lung sections from A, treated with sialidase or buffer alone, then stained with Siglec-F-Fc, Siglec-E-Fc, or CD22-Fc (green); anti-eMBP (red); anti-proSP-C (blue); and DAPI (white). Scale bar represents 50 μm. Results are representative of two independent experiments.

Michael L. Patnode, et al. J Biol Chem. 2013 September 13;288(37):26533-26545.
3.
FIGURE 1.

FIGURE 1. From: Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue.

Siglec-F ligand expression in peripheral blood leukocytes. A, flow cytometry analysis of leukocyte subsets stained with Siglec-F-Fc, Siglec-E-Fc, and CD22-Fc (red histograms). Staining after sialidase treatment (black histograms) and staining with human IgG (gray histograms) are shown. Results are representative of four independent experiments. B, flow cytometry analysis of Siglec-F expression on leukocytes from wild type (black histograms) or Siglec-F KO mice (gray histograms). Results are representative of two independent experiments. Eos, eosinophils; Neut, neutrophils; Baso, basophils; Mono, classical monocytes; NK, natural killer cells; T, T cells; B, B cells; Alv Mac, alveolar macrophages.

Michael L. Patnode, et al. J Biol Chem. 2013 September 13;288(37):26533-26545.
4.
FIGURE 8.

FIGURE 8. From: Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue.

Siglec-F ligand expression in leukocytes from KSGal6ST/C6ST-1 DKO mice. A, flow cytometry analysis of leukocyte subsets stained with Siglec-F-Fc (red histograms). Staining after sialidase treatment (black histograms) and staining with human IgG (gray histograms) are shown. Scale bars represent 50 μm. Results are representative of two independent experiments. B, cryostat-cut sections of lungs from N. brasiliensis-infected mice. Sections were stained with Siglec-F-Fc (green), anti-eMBP (red), anti-proSP-C (blue), and DAPI (white). Low power (top) and high power (bottom) fields are shown. Eos, eosinophils; Neut, neutrophils; Mono, classical monocytes; NK, natural killer cells.

Michael L. Patnode, et al. J Biol Chem. 2013 September 13;288(37):26533-26545.
5.
FIGURE 7.

FIGURE 7. From: Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue.

Siglec-F ligand expression in lungs from KSGal6ST/C6ST-1 DKO mice. A, cryostat-cut sections of lungs from WT or DKO mice stained with Siglec-F-Fc (green), anti-sialoadhesin (red), anti-proSP-C (blue), and DAPI (white). Scale bar represents 50 μm. Results are representative of two independent experiments. B, Siglec-F-Fc reactivity (left) in BAL fluid fractions from WT (filled circles) or DKO mice (filled squares), assayed by ELISA. Results are representative of two independent experiments. The signal was eliminated by sialidase treatment (open circles, open squares). Anti-MUC5B reactivity (right) in BAL fluid fractions from WT (filled circles) and DKO mice (filled squares) was assayed by ELISA. Isotype control signal was minimal (open circles, open squares). Total protein was determined by measuring absorbance at 280 nm for wild type (dotted line) and DKO mice (dashed line). WT, wild type; DKO, KSGal6ST/C6ST-1 double knock-out.

Michael L. Patnode, et al. J Biol Chem. 2013 September 13;288(37):26533-26545.
6.
FIGURE 6.

FIGURE 6. From: Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue.

Analysis of sulfated glycans in BAL fluid and lung KS. A, representative negative ion mode nanoESI HCD MS/MS spectra of mono-sulfated mono-sialylated (top) and mono-sulfated non-sialylated (bottom) O-glycan structures found in BAL fluid from wild type mice. Identification of terminal Gal3S and internal GlcNAc6S was based on previously established diagnostic ions. A weak signal at m/z 167 (bold) may indicate the presence of Gal6S in the non-sialylated structures. Additional clusters of fragment ions around m/z 500–800 are assigned as shown, which are mostly fragment ions resulting from cleavage along the GalNAcitol and consistent with the sulfated LacNAc being extended from the 6-arm. B, reversed-phase ion-pair chromatography analysis of KS from lungs of wild type mice. Standard substances were eluted at the peak positions indicated by arrows. The fragment (6S)Galβ1→4(6S)GlcNAc was not detected in the sample.

Michael L. Patnode, et al. J Biol Chem. 2013 September 13;288(37):26533-26545.
7.
FIGURE 5.

FIGURE 5. From: Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue.

MS analysis of sulfated glycans in eosinophils. A, flow cytometry analysis of IL-5 transgenic eosinophils stained with Siglec-F-Fc (red histogram). Staining after sialidase treatment (black histogram) and staining with human IgG (gray histogram) is shown. Results are representative of three independent experiments. B, extracted ion chromatograms of the major sulfated O-glycans from IL-5 transgenic eosinophils as detected by nanoLC-MS/MS analysis. The m/z values for the [M − H] molecular ions afforded by the mono-sulfated permethylated O-glycans were annotated along with the assigned structures based on interpretation of the HCD and CID MS/MS data. The relative peak heights are indicative of the relative abundance of each of the sulfated, sialylated core 1 and core 2 O-glycans. C, low mass regions of the negative ion mode nanoESI HCD and CID MS/MS spectra of mono-sulfated di-sialylated (left) and mono-sulfated mono-sialylated (right) structures. Assignment of the major peaks for all spectra is annotated using the standard schematic symbols. Eos, eosinophils.

Michael L. Patnode, et al. J Biol Chem. 2013 September 13;288(37):26533-26545.
8.
FIGURE 2.

FIGURE 2. From: Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue.

Siglec-F ligand expression in resident lung cells. A, cryostat-cut sections of lungs from WT mice stained with Siglec-F-Fc (green), anti-sialoadhesin (red), anti-proSP-C (blue), and DAPI (white). Arrowheads mark alveolar macrophages. Arrows mark type II alveolar epithelial cells. The asterisk marks airway epithelium. B, a high power view of the same section shown in A. Siglec-F-Fc staining after sialidase treatment, Siglec-E-Fc staining, and CD22-Fc staining are shown. The inset depicts a type II AEC from a different field (scale bar 10 μm). Results are representative of four independent experiments. C, flow cytometry analysis of Siglec-F-Fc staining of leukocytes from wild type (red histograms) or Siglec-F KO (blue histograms) mice. Sialidase treatment eliminated staining on cells from both Siglec-F KO (gray histograms) and wild type mice (not shown). Results are representative of two independent experiments. Eos, eosinophils; Neut, neutrophils; Alv Mac, alveolar macrophages. Scale bars represent 50 μm unless otherwise stated.

Michael L. Patnode, et al. J Biol Chem. 2013 September 13;288(37):26533-26545.

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