Journal: The Journal of Biological Chemistry

Article Title: Nuclear Receptor Liver X Receptor Is O -GlcNAc-modified in Response to Glucose *

doi: 10.1074/jbc.M109.082685

Figure Lengend Snippet: LXRα and LXRβ are modified by O-GlcNAc in Huh7 cells. A, Huh7 cells transfected with FLAG-hLXRα (α) or FLAG-hLXRβ (β) (25 mm glucose, 2.5% serum) were subjected to SDS-PAGE and blotted using anti-LXRα, anti-LXRβ, or anti-FLAG antibodies. B, FLAG-hLXRα and FLAG-hLXRβ were immunoprecipitated (IP) from Huh7 cells (25 mm glucose, 10% serum) using anti-LXRα or anti-LXRβ antibodies. Input (In, 8%) and immunoprecipitated proteins were subjected to SDS-PAGE and blotted with anti-O-GlcNAc antibodies CTD110.6 or RL2, or sWGA-HRP to verify O-GlcNAc modification. Input shows total amount of O-GlcNAc-modified proteins. Anti-LXRα (left panel), anti-LXRβ (right panel), and anti-FLAG antibodies were used to detect LXRs. C and D, FLAG-hLXRα (C) or FLAG-hLXRβ (D) was immunoprecipitated from Huh7 cells (25 mm glucose, 10% serum) using anti-LXRα or anti-LXRβ antibodies or anti-FLAG M2 affinity gel. Mouse IgG immunoprecipitation was performed as control. Input (8%) and immunoprecipitated proteins were subjected to SDS-PAGE and blotted with anti-LXRα, anti-LXRβ, or anti-O-GlcNAc (RL2) antibodies. Specificity was confirmed by GlcNAc competition (bottom panels).

Article Snippet: Western Blotting O -GlcNAc levels were determined by SDS-PAGE and blotting with mouse monoclonal anti- O - GlcNAc antibodies RL2 (MA1-072, 1:1000; Affinity BioReagents), CTD110.6 (MMS-248R, 1:1000; Covance), or the GlcNAc-binding lectin sWGA conjugated to horseradish peroxidase (HRP), sWGA-HRP (H-2102-1, 1:10,000; EY Laboratories).

Techniques: Modification, Transfection, SDS Page, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Nuclear Receptor Liver X Receptor Is O -GlcNAc-modified in Response to Glucose *

doi: 10.1074/jbc.M109.082685

Figure Lengend Snippet: LXR GlcNAcylation is elevated by glucose in Huh7 cells and in stably transfected FLAG-hLXRα FlpInTM293 cells. A, GlcNAc-modified proteins from FLAG-hLXRα or FLAG-hLXRβ-overexpressing Huh7 cells (1 mm and 25 mm glucose) were absorbed on sWGA-agarose beads (total amount of proteins loaded onto sWGA beads was the same in all lanes). Input (In, 10%) and sWGA-precipitated proteins (sWGA) were subjected to SDS-PAGE and blotted using anti-LXRα or anti-LXRβ antibodies. Immunoprecipitated LXRs are loaded as size control (C) (upper panel). O-GlcNAc-modified proteins from input (10%, left panel) and sWGA precipitation (right panel) were detected using anti-O-GlcNAc (CTD110.6) antibody (lower panel). B, FLAG-hLXRα was immunoprecipitated (IP) from FLAG-hLXRα stably transfected FlpInTM293 cells (5 mm glucose, 25 mm glucose, 5 mm glucose + GlcNAc (NAG), 5 mm glucose + glucosamine) using rabbit anti-FLAG antibody. Input (2%) and immunoprecipitated proteins were subjected to SDS-PAGE and blotted with anti-LXRα antibody and sWGA-HRP or anti-O-GlcNAc (CTD110.6) antibody to verify GlcNAc modification.

Article Snippet: Western Blotting O -GlcNAc levels were determined by SDS-PAGE and blotting with mouse monoclonal anti- O - GlcNAc antibodies RL2 (MA1-072, 1:1000; Affinity BioReagents), CTD110.6 (MMS-248R, 1:1000; Covance), or the GlcNAc-binding lectin sWGA conjugated to horseradish peroxidase (HRP), sWGA-HRP (H-2102-1, 1:10,000; EY Laboratories).

Techniques: Stable Transfection, Transfection, Modification, SDS Page, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Nuclear Receptor Liver X Receptor Is O -GlcNAc-modified in Response to Glucose *

doi: 10.1074/jbc.M109.082685

Figure Lengend Snippet: In vitro GlcNAcylation of LXRα and LXRβ. A, in vitro translated (IVT) 35S-labeled hLXRα or 35S-labeled hLXRβ proteins in rabbit reticulocyte lysate were absorbed on sWGA-agarose beads. Beads were eluted once with 0.5 m galactose (G) followed by three times elution with 0.5 m GlcNAc (E1, E2, E3). In vitro translated lysates (8%) and sWGA eluates (sWGA; E1, E2, E3, G) were subjected to SDS-PAGE and analyzed by fluorescence autoradiography. B, in vitro translated 35S-FLAG-hLXRα or 35S-FLAG-hLXRβ full-length and truncated proteins were absorbed on sWGA-agarose beads. In vitro translated lysates (I, 8%) and sWGA eluates (W) were subjected to SDS-PAGE and analyzed by fluorescence autoradiography. Schematic figures of the LXR proteins are shown. DBD, DNA-binding domain; LBD, ligand-binding domain.

Article Snippet: Western Blotting O -GlcNAc levels were determined by SDS-PAGE and blotting with mouse monoclonal anti- O - GlcNAc antibodies RL2 (MA1-072, 1:1000; Affinity BioReagents), CTD110.6 (MMS-248R, 1:1000; Covance), or the GlcNAc-binding lectin sWGA conjugated to horseradish peroxidase (HRP), sWGA-HRP (H-2102-1, 1:10,000; EY Laboratories).

Techniques: In Vitro, Labeling, SDS Page, Fluorescence, Autoradiography, Binding Assay, Ligand Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Nuclear Receptor Liver X Receptor Is O -GlcNAc-modified in Response to Glucose *

doi: 10.1074/jbc.M109.082685

Figure Lengend Snippet: In vivo hepatic LXR O-GlcNAcylation is induced by refeeding and STZ treatment. A, mice were fasted for 24 h (F) or fasted for 24 h then refed for 12 h (R) (n = 2/lane). B, mice were treated with STZ for 1 week before they were fasted for 24 h then refed for 12 h (STZ-Refed). Controls (Refed) were not treated with STZ (n = 2/lane). A and B, LXRα was immunoprecipitated (IP) using anti-LXR antibody. Input (In, 12%) and immunoprecipitated proteins were subjected to SDS-PAGE and blotted with anti-LXR or sWGA-HRP. Specificity was confirmed by GlcNAc competition (A). Plasma glucose and insulin levels for each animal are shown in the bottom panel. C, hepatic gene expression of SREBP-1c was analyzed by quantitative reverse transcription-PCR and normalized against expression of the ribosomal protein 36B4 (n = 4/group for fasted (gray bar), n = 4–6/group for refed (black bars)). Values are given as mean ± S.D. (error bars), and the expression in the fasted controls is set as 1. **, p < 0.01 STZ versus control; #, p < 0.01 fasted versus refed.

Article Snippet: Western Blotting O -GlcNAc levels were determined by SDS-PAGE and blotting with mouse monoclonal anti- O - GlcNAc antibodies RL2 (MA1-072, 1:1000; Affinity BioReagents), CTD110.6 (MMS-248R, 1:1000; Covance), or the GlcNAc-binding lectin sWGA conjugated to horseradish peroxidase (HRP), sWGA-HRP (H-2102-1, 1:10,000; EY Laboratories).

Techniques: In Vivo, Immunoprecipitation, SDS Page, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Nuclear Receptor Liver X Receptor Is O -GlcNAc-modified in Response to Glucose *

doi: 10.1074/jbc.M109.082685

Figure Lengend Snippet: O-GlcNAc regulates LXR transactivation of the SREBP-1c promoter. A, Huh7 cells were transfected with pBP1c550-Luc reporter, Renilla luciferase control plasmid (pRL), and RXRα expression vector together with LXRα (black bars), LXRβ (gray bars), or empty vector pSG5 (white bars). 5 h after transfection, cells were stimulated with different glucose concentrations (1, 5, or 25 mm) for 24 h. A schematic figure of the reporter construct including LXRE elements a and b is shown. B, Huh7 cells were transfected with different pBP1cLXRE-Luc reporter constructs (LXREab, LXREaM, LXREbM, or LXREabM), pRL LXRα, and RXRα expression vectors. 5 h after transfection, cells were stimulated with different glucose concentrations (1, 5, or 25 mm) for 24 h. A schematic figure of the LXRE reporter constructs is shown. C, Huh7 cells were transfected with pBP1cLXREab-Luc reporter, pRL LXRα, and RXRα expression vectors. 5 h after transfection, cells were stimulated with 5 mm glucose in the absence or presence of PUGNAc (50 μm and 100 μm) for 24 h. The luciferase value at 5 mm glucose without stimulation is set as 1. D, Huh7 cells were transfected with pBP1cLXREab-Luc reporter, pRL LXRα, and RXRα expression vectors. 5 h after transfection, cells were stimulated with 25 mm glucose in the absence or presence of DON (5 μm) and glucosamine (0.2 mm and 1 mm) for 24 h. The luciferase value at 25 mm glucose without stimulation is set as 1. All luciferase data are presented as one representative experiment of three or more independent experiments performed in triplicate ± S.D. (error bars). *, p < 0.05; **, p < 0.01. E, LXRα was immunoprecipitated (IP) from FLAG-hLXRα-overexpressing Huh7 cells treated with 5 mm glucose for 24 h in the absence or presence of 2 mm glucosamine or 100 μm PUGNAc. Immunoprecipitated proteins were subjected to SDS-PAGE and blotted with anti-O-GlcNAc (RL2) or anti-FLAG antibodies. F, O-GlcNAc-modified proteins from FLAG-LXRα-overexpressing Huh7 cells cultured for 24 h in 25 mm d-glucose in the absence or presence of DON (5 μm) were absorbed on sWGA-agarose beads. Input (In, 10%) and sWGA pulled-down proteins (sWGA) were subjected to SDS-PAGE and blotted with anti-LXRα antibody.

Article Snippet: Western Blotting O -GlcNAc levels were determined by SDS-PAGE and blotting with mouse monoclonal anti- O - GlcNAc antibodies RL2 (MA1-072, 1:1000; Affinity BioReagents), CTD110.6 (MMS-248R, 1:1000; Covance), or the GlcNAc-binding lectin sWGA conjugated to horseradish peroxidase (HRP), sWGA-HRP (H-2102-1, 1:10,000; EY Laboratories).

Techniques: Transfection, Luciferase, Plasmid Preparation, Expressing, Construct, Immunoprecipitation, SDS Page, Modification, Cell Culture