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: PerCP-mouse IgG1, PerCP-mouse IgG anti-human CD4 (clone SK3), PerCP-mouse IgG anti-human CD8 (SK1), allophycocyanin-goat anti-human Fc, mouse anti-PSGL-1 mAb KPL-1, and anti-β-actin mAb AC-15 were purchased from BD Biosciences.

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: PerCP-mouse IgG1, PerCP-mouse IgG anti-human CD4 (clone SK3), PerCP-mouse IgG anti-human CD8 (SK1), allophycocyanin-goat anti-human Fc, mouse anti-PSGL-1 mAb KPL-1, and anti-β-actin mAb AC-15 were purchased from BD Biosciences.

Techniques: Expressing, Western Blot, Staining, Flow Cytometry

Journal: Frontiers in molecular biosciences

Article Title: Ciliary phenotyping in renal epithelial cells in a cranioectodermal dysplasia patient with WDR35 variants.

doi: 10.3389/fmolb.2023.1285790

Figure Lengend Snippet: FIGURE 2 Renal epithelial cells (URECs) derived from control 1 and CED patient with WDR35 variants presenting a typical cobblestone epithelial cell layer when grown in 2D culture (A). Human URECs following IF imaging using anti-ARL13B (green) for the ciliary membrane, anti-acetylated-tubulin (red) for the ciliary axoneme, PCNT (pink) to mark the base of the cilium, and DAPI (blue) for the nuclei are shown. P, CED patient; C1, control 1; C2, control 2; and C3, control 3 (B). The IF experiments revealed no significant differences in ciliogenesis between the CED patient and the combined controls (C). There was, however, a statistical difference (p < 0.01) between the CED patient and control 1, a sex- and age-matched control (D).

Article Snippet: After fixation with 2% PFA (Sigma), permeabilization with 1% Triton, and blocking with 2% BSA (Sigma), the cells were incubated with primary antibodies targeting acetylated-alpha-tubulin (Acet-Tub) at 1:1000 (mouse monoclonal T6793, Sigma-Aldrich), ADP-ribosylation factor-like protein 13B (ARL13B) at 1:500 (rabbit polyclonal, Proteintech Group), and pericentrin (PCNT) at 1:1000 (mouse monoclonal, Abcam Cambridge), followed by incubation with fluor-labeled secondary antibodies: anti-mouse Alexa Fluor 568, anti-rabbit Alexa Fluor 488, and anti-mouse Alexa Fluor 647 from Thermo Fisher Scientific.

Techniques: Derivative Assay, Control, Imaging, Membrane

Journal: Frontiers in molecular biosciences

Article Title: Ciliary phenotyping in renal epithelial cells in a cranioectodermal dysplasia patient with WDR35 variants.

doi: 10.3389/fmolb.2023.1285790

Figure Lengend Snippet: FIGURE 3 Cilium length, height, and width were analyzed using two antibodies specific for Acet-Tub and ARL13B. Examination of the cilia width (A axis) revealed highly significant differences between the CED patient and controls for both markers (Acet-Tub and ARL13B: p < 0.0001) (A, B). Evaluation of the ciliary height showed no differences between the CED patient and controls (C, D). Analysis of the ciliary length revealed longer cilia in the CED patient as compared to controls for both proteins (Acet-Tub: p < 0.01 and ARL13B: p < 0.05) (E, F).

Article Snippet: After fixation with 2% PFA (Sigma), permeabilization with 1% Triton, and blocking with 2% BSA (Sigma), the cells were incubated with primary antibodies targeting acetylated-alpha-tubulin (Acet-Tub) at 1:1000 (mouse monoclonal T6793, Sigma-Aldrich), ADP-ribosylation factor-like protein 13B (ARL13B) at 1:500 (rabbit polyclonal, Proteintech Group), and pericentrin (PCNT) at 1:1000 (mouse monoclonal, Abcam Cambridge), followed by incubation with fluor-labeled secondary antibodies: anti-mouse Alexa Fluor 568, anti-rabbit Alexa Fluor 488, and anti-mouse Alexa Fluor 647 from Thermo Fisher Scientific.

Techniques:

Journal: Frontiers in molecular biosciences

Article Title: Ciliary phenotyping in renal epithelial cells in a cranioectodermal dysplasia patient with WDR35 variants.

doi: 10.3389/fmolb.2023.1285790

Figure Lengend Snippet: FIGURE 4 Volume measurements of ARL13B, Acet-Tub, and PCNT. The amount of all markers was calculated and revealed a highly significant increase in volume for all three proteins (Acet-Tub, ARL13B, and PCNT: p < 0.0001) (A–C).

Article Snippet: After fixation with 2% PFA (Sigma), permeabilization with 1% Triton, and blocking with 2% BSA (Sigma), the cells were incubated with primary antibodies targeting acetylated-alpha-tubulin (Acet-Tub) at 1:1000 (mouse monoclonal T6793, Sigma-Aldrich), ADP-ribosylation factor-like protein 13B (ARL13B) at 1:500 (rabbit polyclonal, Proteintech Group), and pericentrin (PCNT) at 1:1000 (mouse monoclonal, Abcam Cambridge), followed by incubation with fluor-labeled secondary antibodies: anti-mouse Alexa Fluor 568, anti-rabbit Alexa Fluor 488, and anti-mouse Alexa Fluor 647 from Thermo Fisher Scientific.

Techniques: