Journal: iScience

Article Title: GFAT2 mediates cardiac hypertrophy through HBP-O-GlcNAcylation-Akt pathway

doi: 10.1016/j.isci.2021.103517

Figure Lengend Snippet: Key resources table

Article Snippet: Rabbit polyclonal anti-OGA , Novus Biologicals , Cat#NBP1-81244; RRID: AB_11053819.

Techniques: Plasmid Preparation, Virus, Recombinant, Sequencing, Software, Microscopy

Journal: Genome Research

Article Title: Prepatterning of differentiation-driven nuclear lamin A/C-associated chromatin domains by GlcNAcylated histone H2B

doi: 10.1101/gr.193748.115

Figure Lengend Snippet: Assessment of specificity of the anti-H2BS112GlcNAc antibody. ( A ) Immunoreactivity of the anti-H2BS112GlcNAc antibody on H2BS112GlcNAc and corresponding unmodified H2B peptides (KHAVS 112 EGTK) immobilized on nitrocellulose. Peptides were preincubated without or with 2 ng/µL recombinant OGA prior to immunodetection. ( B ) Immunofluorescence peptide competition assay using H2BS112GlcNAc and unmodified H2B peptides; immunodetection using anti-H2BS112GlcNAc. Scale bar, 10 μm. ( C ) Immunoblot of ASC extracts preincubated with 0 or 2 ng/µL recombinant OGA for 30 min. ( D ) Expression of an EGFP-H2BS112A mutant, but not a control wild-type EGFP-H2B, in HeLa cells reduces H2BS112GlcNAc immunoreactivity ( left ) without affecting detection of total H2B ( middle ) or EGFP ( right ).

Article Snippet: Lysates were incubated for 30 min at room temperature with 2 ng/μL recombinant OGA (6779GH020, R&D Systems) and dissolved in 2× Laemmli buffer for SDS-PAGE and immunoblotting.

Techniques: Recombinant, Immunodetection, Immunofluorescence, Competitive Binding Assay, Western Blot, Expressing, Mutagenesis, Control

Journal: BMC molecular and cell biology

Article Title: Sex alters thyroid hormone's effect on protein O-GlcNAcylation in the aged mouse heart.

doi: 10.1186/s12860-025-00543-x

Figure Lengend Snippet: Fig. 1 The hexosamine biosynthesis pathway and O-GlcNAcylation. GFAT, L-glutamine: fructose-6-phosphate amidotransferase; OGT, O-GlcNAc transferase; OGA, O-GlcNAcase; UDP-GlcNAc, UDP-N-acetylglucosamine; NAGK, N acetyl-glucosamine kinase

Article Snippet: The primary antibodies used in this study were as follows: anti-O-linked N-acetylglucosamine [RL2] (#ab2730, Abcam, Cambridge, UK), OGT(#ab96718, Abcam, Cambridge, UK), OGA (#NBP1-81244, Novus Biologicals, Centennial, CO), GFAT1 (#NBP3-04455, Novus Biologicals, Centennial, CO), GFAT2 (#sc-134710, Santa Cruz Biotechnologies, Dallas, TX) and NAGK (#15051-1-AP, Proteintech, Rosemont, IL).The secondary antibodies used in this study was goat anti-mouse IgG, light chain specific (#115-035-174, Jackson ImmunoResearch, West Grove PA) and goat anti-rabbit IgG (H + L) (#1706515, Bio-Rad, Hercules, CA).

Techniques:

Journal: BMC molecular and cell biology

Article Title: Sex alters thyroid hormone's effect on protein O-GlcNAcylation in the aged mouse heart.

doi: 10.1186/s12860-025-00543-x

Figure Lengend Snippet: Fig. 11 ACAA2/TRβ1 transcriptional assay. Inhibiting O-GlcNAc in transfected TH-treated CV-1 cells reduced ACAA2 activation of TRβ1 transcriptional activity. TRβ1 – thyroid receptor β1; ACAA2 – acetyl-coenzyme A acyltransferase; T3 – thyroid hormone; TG – thiamet G, OGA inhibitor; OSMI-1 – OGT inhibitor. Statistics – one-way ANOVA – correct for multiple comparison- test: Šidák post hoc analysis was performed (n = 4).

Article Snippet: The primary antibodies used in this study were as follows: anti-O-linked N-acetylglucosamine [RL2] (#ab2730, Abcam, Cambridge, UK), OGT(#ab96718, Abcam, Cambridge, UK), OGA (#NBP1-81244, Novus Biologicals, Centennial, CO), GFAT1 (#NBP3-04455, Novus Biologicals, Centennial, CO), GFAT2 (#sc-134710, Santa Cruz Biotechnologies, Dallas, TX) and NAGK (#15051-1-AP, Proteintech, Rosemont, IL).The secondary antibodies used in this study was goat anti-mouse IgG, light chain specific (#115-035-174, Jackson ImmunoResearch, West Grove PA) and goat anti-rabbit IgG (H + L) (#1706515, Bio-Rad, Hercules, CA).

Techniques: Transcription Assay, Transfection, Activation Assay, Activity Assay, Comparison

Journal: Frontiers in Immunology

Article Title: O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis

doi: 10.3389/fimmu.2024.1387197

Figure Lengend Snippet: Hexosamine biosynthetic pathway genes are upregulated in IPF fibroblasts and myofibroblasts. (A) Schematic of simplified HBP and OGT/OGA cycling of O-GlcNAc. Image created with Biorender. (B) Cluster map of cell types: pericytes, smooth muscle cells, fibroblasts, and myofibroblasts, expressing CTHRC1 from IPF (n=32) and non-IPF (n=29) donors. (C) Spatial mapping of fibroblasts and myofibroblasts expressing HBP and profibrotic genes based on scaled expression, gray coloration denotes no expression. (D–J) Transcript expression of HBP genes and profibrotic genes from 5 IPF donors and 5 non-IPF primary human lung fibroblasts. Data from multiple replicates are presented as mean ± SEM, each dot represents biological replicates averaged from three technical replicates. Outliers determined by the 1.5xIQR rule were excluded, and statistical analyses were done using the Student’s t-test. *p<0.05, **p<0.01. GFAT, Glutamine fructose-6-phosphate aminotransferase; GFPT1 , glutamine fructose-6-phosphate transaminase 1; O-GlcNAc, O-linked N-Acetylglucosamine; OGT, O-GlcNAc transferase; OGA, O-GlcNAc hydrolase; MGEA5 , meningioma expressed antigen 5; ACTA2 , actin, alpha 2; smooth muscle.

Article Snippet: Primary antibodies used in this study: Pan Collagen (1:800; cat no: PA5-104252, Thermo Fisher Scientific), Col1α1 [EPR22894-89] (1:1000; cat no: ab260043; abcam), Col3α1 (1:1000; cat no: A3795; Abclonal), Pericardin (1:1000; cat no: EC11; Developmental Biology Hybridoma Bank), OGT (1:1000; cat no: GTX109939, Genetex), OGA (1:1000; cat no: A304-345A; Bethyl Lab), Smad3 (1:1000; cat no: 9523S; Cell Signaling), S-tag (1:1000; cat no: 12774S; Cell Signaling), β-actin-HRP (1:10,000; cat no: A3854-200UL; Millapore Sigma), α-Tubulin (1:10,000; cat no: 12G10; Developmental Biology Hybridoma Bank).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis

doi: 10.3389/fimmu.2024.1387197

Figure Lengend Snippet: O-GlcNAc modified proteins identified by mass spectrometry involved in TGF-β signaling and fibrosis.

Article Snippet: Primary antibodies used in this study: Pan Collagen (1:800; cat no: PA5-104252, Thermo Fisher Scientific), Col1α1 [EPR22894-89] (1:1000; cat no: ab260043; abcam), Col3α1 (1:1000; cat no: A3795; Abclonal), Pericardin (1:1000; cat no: EC11; Developmental Biology Hybridoma Bank), OGT (1:1000; cat no: GTX109939, Genetex), OGA (1:1000; cat no: A304-345A; Bethyl Lab), Smad3 (1:1000; cat no: 9523S; Cell Signaling), S-tag (1:1000; cat no: 12774S; Cell Signaling), β-actin-HRP (1:10,000; cat no: A3854-200UL; Millapore Sigma), α-Tubulin (1:10,000; cat no: 12G10; Developmental Biology Hybridoma Bank).

Techniques: Modification, Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis

doi: 10.3389/fimmu.2024.1387197

Figure Lengend Snippet: Hexosamine biosynthetic pathway genes are upregulated in IPF fibroblasts and myofibroblasts. (A) Schematic of simplified HBP and OGT/OGA cycling of O-GlcNAc. Image created with Biorender. (B) Cluster map of cell types: pericytes, smooth muscle cells, fibroblasts, and myofibroblasts, expressing CTHRC1 from IPF (n=32) and non-IPF (n=29) donors. (C) Spatial mapping of fibroblasts and myofibroblasts expressing HBP and profibrotic genes based on scaled expression, gray coloration denotes no expression. (D–J) Transcript expression of HBP genes and profibrotic genes from 5 IPF donors and 5 non-IPF primary human lung fibroblasts. Data from multiple replicates are presented as mean ± SEM, each dot represents biological replicates averaged from three technical replicates. Outliers determined by the 1.5xIQR rule were excluded, and statistical analyses were done using the Student’s t-test. *p<0.05, **p<0.01. GFAT, Glutamine fructose-6-phosphate aminotransferase; GFPT1 , glutamine fructose-6-phosphate transaminase 1; O-GlcNAc, O-linked N-Acetylglucosamine; OGT, O-GlcNAc transferase; OGA, O-GlcNAc hydrolase; MGEA5 , meningioma expressed antigen 5; ACTA2 , actin, alpha 2; smooth muscle.

Article Snippet: For gene expression analysis, 10 ng of cDNA was used for quantitative PCR (qPCR) (StepOnePlus Real-Time PCR; Thermo Fisher Scientific) with the following Taqman probes (Life Technologies/Applied Biosystems): GAPDH (Hs02758991; Mm99999915_g1), OGT (Hs00269228_m1; Mm00507317_m1), MGEA5 (Hs00201970_m1; Mm00452409_m1), GFPT1 (Hs00899865_m1), (Hs01049570_m1), MM9 (Mm00442991_m1), COL1A1 (Hs00164004_m1; Mm01302043_g1), COL3A1 (Hs00943809_m1; Mm00802331_m1), ACTA2 (Hs00426835_g1; Mm01546133_m1), TGFB1 (Hs00998133_m1; Mm01178820_m1), MMP2 (Mm00439498_m1), TIMP3 (Mm00441826_m1).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis

doi: 10.3389/fimmu.2024.1387197

Figure Lengend Snippet: O-GlcNAc modified proteins identified by mass spectrometry involved in TGF-β signaling and fibrosis.

Article Snippet: For gene expression analysis, 10 ng of cDNA was used for quantitative PCR (qPCR) (StepOnePlus Real-Time PCR; Thermo Fisher Scientific) with the following Taqman probes (Life Technologies/Applied Biosystems): GAPDH (Hs02758991; Mm99999915_g1), OGT (Hs00269228_m1; Mm00507317_m1), MGEA5 (Hs00201970_m1; Mm00452409_m1), GFPT1 (Hs00899865_m1), (Hs01049570_m1), MM9 (Mm00442991_m1), COL1A1 (Hs00164004_m1; Mm01302043_g1), COL3A1 (Hs00943809_m1; Mm00802331_m1), ACTA2 (Hs00426835_g1; Mm01546133_m1), TGFB1 (Hs00998133_m1; Mm01178820_m1), MMP2 (Mm00439498_m1), TIMP3 (Mm00441826_m1).

Techniques: Modification, Mass Spectrometry