Journal: The Journal of Biological Chemistry

Article Title: Peracetylated 4-Fluoro-glucosamine Reduces the Content and Repertoire of N - and O -Glycans without Direct Incorporation *

doi: 10.1074/jbc.M110.194597

Figure Lengend Snippet: Measurement of UDP-GlcNAc level in extracts of 4-F-GlcNAc-treated sLe X (+) KG1a and T cells. a , chair configurations of 4-F-GlcNAc (peracetylated) and structurally related control analogs, UDP-GlcNAc, GlcNAc, α-Glc pentaacetate, and 4-F-α-GlcNAc (unacetylated). b , assay specificity for detecting the sugars shown in a. c and d , level of UDP-GlcNAc measured in sLe X (+) KG1a or T cell extracts. KG1a cells were incubated for 48 h with 0.05 m m sugars. T cells were incubated for 48 h with 0.05 m m sugars or for 38 h with 0.01–0.025 m m 4-F-GlcNAc (peracetylated). Shown is mean UDP-GlcNAc level with S.E. indicated when compared with untreated control. Experiments were performed in triplicate on at least three separate occasions and donors. Statistically significant differences compared with untreated controls are shown as follows. *, p < 0.05; **, p < 0.01; ***, p < 0.001, one-way analysis of variance with Dunnett's post hoc test.

Article Snippet: Anti-sLe X mAb (HECA-452) was purchased from BD PharMingen, Inc. (San Diego, CA).

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: Peracetylated 4-Fluoro-glucosamine Reduces the Content and Repertoire of N - and O -Glycans without Direct Incorporation *

doi: 10.1074/jbc.M110.194597

Figure Lengend Snippet: Analysis of 4-F-GlcNAc efficacy on E-selectin and Gal-1 ligand expression on sLe X KG1a cells. a , Western blots of KG1a cells treated with diluent control ( Untreated ), 0.05 m m 4-F-GlcNAc, or 4-F-GlcNAc and bromelain were stained with mouse E-selectin-human IgG Fc1 ( E-sel.-hFc ) (1 μg/ml), Gal-1hFc (10 μg/ml), anti-sLe X mAb (1 μg/ml), anti-PSGL-1 mAb (1 μg/ml), or anti-β-actin mAb (1 μg/ml). b , using NIH ImageJ, densitometric gray scale units of scanned lanes from 50 to 260 kDa of triplicate Western blots were measured and plotted as mean ± S.D. ( error bars ) in relative gray units. c , flow cytometry of KG1a cells treated with diluent control ( Untreated ), 0.05 m m 4-F-GlcNAc, or 4-F-GlcNAc and bromelain were stained with mouse E-selectin-human IgG Fc1 (1 μg/ml), Gal-1hFc (10 μg/ml), anti-sLe X mAb (1 μg/ml), anti-PSGL-1 mAb (1 μg/ml), or respective isotype control and respective fluorophore-conjugated secondary antibody. d , mean fluorescent intensities from triplicate flow cytometry experiments were compared with untreated control and presented as percentage of untreated control. Statistically significant differences when compared with untreated controls are shown as follows. **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti-sLe X mAb (HECA-452) was purchased from BD PharMingen, Inc. (San Diego, CA).

Techniques: Expressing, Western Blot, Staining, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Peracetylated 4-Fluoro-glucosamine Reduces the Content and Repertoire of N - and O -Glycans without Direct Incorporation *

doi: 10.1074/jbc.M110.194597

Figure Lengend Snippet: Analysis of 4-F-GlcNAc efficacy on E-selectin and Gal-1 ligand expression on sLe X (+) T cells. a , Western blots of T cells treated with diluent control ( Untreated ), 0.05 m m 4-F-GlcNAc, or 4-F-GlcNAc and bromelain were stained with mouse E-selectin-human IgG Fc1 ( E-sel.-hFc ) (1 μg/ml), Gal-1hFc (10 μg/ml), anti-sLe X mAb (1 μg/ml), anti-PSGL-1 mAb (1 μg/ml), or anti-β-actin mAb (1 μg/ml). b , using NIH ImageJ, densitometric gray scale units of scanned lanes from 50 to 260 kDa of triplicate Western blots were measured and plotted as mean ± S.D. ( error bars ) in relative gray units. c , flow cytometry of T cells treated with diluent control, 0.05 m m 4-F-GlcNAc, or 4-F-GlcNAc and bromelain were stained with mouse E-selectin-human IgG Fc1 (1 μg/ml), Gal-1hFc (10 μg/ml), anti-sLe X mAb (1 μg/ml), anti-PSGL-1 mAb (1 μg/ml), or the respective isotype control and respective fluorophore-conjugated secondary antibody. d , mean fluorescent intensities from triplicate flow cytometry experiments were compared with untreated control and presented as percentage of untreated control. Statistically significant differences when compared with untreated controls are shown as follows. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti-sLe X mAb (HECA-452) was purchased from BD PharMingen, Inc. (San Diego, CA).

Techniques: Expressing, Western Blot, Staining, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Peracetylated 4-Fluoro-glucosamine Reduces the Content and Repertoire of N - and O -Glycans without Direct Incorporation *

doi: 10.1074/jbc.M110.194597

Figure Lengend Snippet: Gene expression analysis of 4-F-GlcNAc-treated sLe X (+) KG1a and T cells. a , heat maps of differentially expressed gene transcripts from untreated versus 4-F-GlcNAc-treated KG1a cells and untreated versus 4-F-GlcNAc-treated T cells depict increased ( red ) and decreased ( blue ) expression relative to the mean transcript expression values. At FC of 4-F-GlcNAc-treated/untreated ratios greater than 1.4, there were 17 and 94 differentially expressed gene transcripts from KG1a and T cells, respectively (adjusted p value of <0.1). b , gene expression clustering from untreated and 4-F-GlcNAc-treated KG1a and T cells of triplicate experiments was validated using centered correlation and average linkage and is represented in a dendrogram. c , a Venn diagram demonstrated that five genes were similarly modulated in KG1a and T cells (adjusted p value of <0.15).

Article Snippet: Anti-sLe X mAb (HECA-452) was purchased from BD PharMingen, Inc. (San Diego, CA).

Techniques: Expressing