Journal: Glycobiology

Article Title: Detection of cardiac O-GlcNAcylation via subcellular fractionation and dual antibody analysis in pressure overload cardiac hypertrophy

doi: 10.1093/glycob/cwaf086

Figure Lengend Snippet: Global unfractionated cardiac protein O-GlcNAc levels detected by RL2 or CTD110.6 antibodies. Males (a) and females (b) comparing sham, 1wTAC and 6wTAC. All values are arbitrary units (a.U.) normalized to total protein levels. Statistical significance was based on unpaired t -test with a Bonferroni correction ( P < 0.017). Brackets denote significance between indicated groups. ◂, κ (kappa) light chains IgG. * , significant band difference between the sham and 6wTAC ( P = 0.014).

Article Snippet: From Novus Biologicals, Centennial, CO: O-GlcNAc RL2 (catalog # NB300–524).

Techniques:

Journal: Glycobiology

Article Title: Detection of cardiac O-GlcNAcylation via subcellular fractionation and dual antibody analysis in pressure overload cardiac hypertrophy

doi: 10.1093/glycob/cwaf086

Figure Lengend Snippet: Male to female comparison of global unfractionated cardiac protein O-GlcNAc levels detected by RL2 and CTD110.6. (a) Sham, (b) 1wTAC, and (c) 6wTAC. All values are arbitrary units (a.U.) normalized to total protein levels. Statistical significance was based on unpaired t -test ( P < 0.05). Brackets denote significance between indicated groups. ◂, κ (kappa) light chains IgG.

Article Snippet: From Novus Biologicals, Centennial, CO: O-GlcNAc RL2 (catalog # NB300–524).

Techniques: Comparison

Journal: Glycobiology

Article Title: Detection of cardiac O-GlcNAcylation via subcellular fractionation and dual antibody analysis in pressure overload cardiac hypertrophy

doi: 10.1093/glycob/cwaf086

Figure Lengend Snippet: O-GlcNAc levels in the cardiac protein cytoplasmic fraction detected by RL2 and CTD110.6. Males (a) and females (b) comparing sham, 1wTAC and 6wTAC. All values are arbitrary units (a.U.) normalized to total protein levels. Statistical significance was based on unpaired t -test with a Bonferroni correction ( P < 0.017). Brackets denote significance between indicated groups. ◂, κ (kappa) light chains IgG.

Article Snippet: From Novus Biologicals, Centennial, CO: O-GlcNAc RL2 (catalog # NB300–524).

Techniques:

Journal: Glycobiology

Article Title: Detection of cardiac O-GlcNAcylation via subcellular fractionation and dual antibody analysis in pressure overload cardiac hypertrophy

doi: 10.1093/glycob/cwaf086

Figure Lengend Snippet: O-GlcNAc levels in the cardiac protein insoluble fraction detected by RL2 and CTD110.6. Males (a) and females (b) comparing sham, 1wTAC and 6wTAC. All values are arbitrary units (a.U.) normalized to total protein levels. Statistical significance was based on unpaired t -test with a Bonferroni correction ( P < 0.017). Brackets denote significance between indicated groups.

Article Snippet: From Novus Biologicals, Centennial, CO: O-GlcNAc RL2 (catalog # NB300–524).

Techniques:

Journal: Glycobiology

Article Title: Detection of cardiac O-GlcNAcylation via subcellular fractionation and dual antibody analysis in pressure overload cardiac hypertrophy

doi: 10.1093/glycob/cwaf086

Figure Lengend Snippet: O-GlcNAc levels in the cardiac protein membrane fraction detected by RL2 and CTD110.6. Males (a) and females (b) comparing sham, 1wTAC and 6wTAC. All values are arbitrary units (a.U.) normalized to total protein levels. Statistical significance was based on unpaired t -test with a Bonferroni correction ( P < 0.017). Brackets denote significance between indicated groups. ◂, κ (kappa) light chains IgG.

Article Snippet: From Novus Biologicals, Centennial, CO: O-GlcNAc RL2 (catalog # NB300–524).

Techniques: Membrane

Journal: Glycobiology

Article Title: Detection of cardiac O-GlcNAcylation via subcellular fractionation and dual antibody analysis in pressure overload cardiac hypertrophy

doi: 10.1093/glycob/cwaf086

Figure Lengend Snippet: O-GlcNAc levels in the cardiac protein soluble nuclear fraction detected by RL2 and CTD110.6. Males (a) and females (b) comparing sham, 1wTAC and 6wTAC. All values are arbitrary units (a.U.) normalized to total protein levels. Statistical significance was based on unpaired t -test with a Bonferroni correction ( P < 0.017). Brackets denote significance between indicated groups.

Article Snippet: From Novus Biologicals, Centennial, CO: O-GlcNAc RL2 (catalog # NB300–524).

Techniques:

Journal: bioRxiv

Article Title: Mapping the endothelial O-GlcNAcome uncovers CCAR1 as a regulator of senescence

doi: 10.64898/2026.02.12.705616

Figure Lengend Snippet: (A) HUVEC were repeatedly transfected with non-targeting siRNA (siControl) or co-transfected with GFAT1- and OGT-specific (siGFAT1+OGT) siRNAs (96h, twice) and seeded in full growth medium for BrdU assay; after 24 h, medium was changed for low serum (2% FCS) medium and after additional 4 h, cells were either treated with bFGF (50 ng/ml, 24h) or left unstimulated. (B, G) Spheroids generated from HUVEC depleted for O-GlcNAc using either genetic (B) or pharmacological (G) approach were treated with 10 or 50 ng/ml VEGF, respectively, or left unstimulated. Representative images of spheroids (B, G) and analysis of the number of sprouts per spheroid (C, D, H), mean sprout lengths (E) and sum of sprout lengths (F) are shown. Data are means ± SEM of 4 independent experiments using cells from different donors. *p < 0.05, **p < 0.01, ***p < 0.001 vs. respective unstimulated control (-); # p < 0.05 vs. control siRNA or diluent control ( H ). Two-way ANOVA with Bonferroni’s multiple comparison test (A, C, H) , or paired t-test (D-F) were used.

Article Snippet: The following antibodies were purchased from Cell Signalling Technology: O-GlcNAc (CTD110.6 clone, #9875), OGT (#24083), p16 INK4A (#92803), p21 Cip1 (#2947), phospho(T202/Y204)-Erk1/2 (#9106), Erk1/2 (#9107), phospho(S139)-H2A.X (#9718), phospho(S15)-p53 (#92845), phospho(S46)-p53 (#2521), phospho(S780)-Rb (#9307), β-actin (#4970); caspase-3 (#9665), cleaved caspase-3 (#9664), PTMScan ® O-GlcNAc [GlcNAc-S/T] Motif Kit (#95220).

Techniques: Transfection, BrdU Staining, Generated, Control, Comparison

Journal: bioRxiv

Article Title: Mapping the endothelial O-GlcNAcome uncovers CCAR1 as a regulator of senescence

doi: 10.64898/2026.02.12.705616

Figure Lengend Snippet: (A) Human umbilical vein endothelial cell (HUVEC) lysates (Thiamet G-treated) were subjected to O-GlcNAc peptide-level enrichment using an antibody mix. (B) Overlap of unique HexNAc peptide and corresponding protein identifications from biological triplicates (Glycan FDR < 0.05). (C) HexNAc-modified proteins detected in at least two of three replicates (N = 187), overlapping with senescence-associated proteins from the SeneQuest database (filtered for proteins up- or downregulated in ≥3 studies, N = 893). The protein-protein interaction network was visualized using STRING with MCL clustering. (D) Gene Ontology (GO) enrichment for Molecular Function of HexNAc-modified proteins identified in at least two of three replicates (N = 187), shown in a simplified form. (E) Lysates from basal or O-GlcNAc-enriched HUVEC (Thiamet G-treated vs. untreated) were immunoprecipitated using the RL2 antibody or a control IgG and analyzed by LC-MS. (F) Venn diagram showing proteins identified in all three biological replicates, excluding identifications from the IgG control IP. (G) Gene Ontology (GO) enrichment for Molecular Function of HexNAc- modified proteins identified in at least two of three replicates (N = 187), shown in a simplified form. (H) Mean intensities (max-LFQ, n=3) of immunoprecipitated proteins comparing Thiamet G treatment vs. vehicle control (untreated). Only proteins quantified in all three replicates in both groups are shown.

Article Snippet: The following antibodies were purchased from Cell Signalling Technology: O-GlcNAc (CTD110.6 clone, #9875), OGT (#24083), p16 INK4A (#92803), p21 Cip1 (#2947), phospho(T202/Y204)-Erk1/2 (#9106), Erk1/2 (#9107), phospho(S139)-H2A.X (#9718), phospho(S15)-p53 (#92845), phospho(S46)-p53 (#2521), phospho(S780)-Rb (#9307), β-actin (#4970); caspase-3 (#9665), cleaved caspase-3 (#9664), PTMScan ® O-GlcNAc [GlcNAc-S/T] Motif Kit (#95220).

Techniques: Glycoproteomics, Modification, Immunoprecipitation, Control, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Mapping the endothelial O-GlcNAcome uncovers CCAR1 as a regulator of senescence

doi: 10.64898/2026.02.12.705616

Figure Lengend Snippet: O-GlcNAc-enriched tryptic peptides (N=3) were measured by LC-TIMS/MS with HCD fragmentation. The analysis was performed on the PSM level. (A) Distribution of unmodified and HexNAc-modified PSMs in the ion mobility (1/K0) vs. m/z dimension. Shown are unique peptide sequences for each charge state (Unmodified: N = 23,157 HexNAc: N = 505). (B) Relative abundance of peptide misscleavages for unmodified and HexNAc-modified PSMs. (C-E) Unmodified and HexNAc-modified PSMs were compared. Shown are unique peptide sequences, with the highest observed intensity (Unmodified: N = 18,73867; O-GlcNAc: N = 416). (C) Peptide length distribution. (D) Retention time distribution. (E) Distribution of the GRAVY score as a metric of hydrophobicity, with a higher score indicating higher hydrophobicity. Asymptotic two-sample Kolmogorov-Smirnov-test, **** p<1e-04.

Article Snippet: The following antibodies were purchased from Cell Signalling Technology: O-GlcNAc (CTD110.6 clone, #9875), OGT (#24083), p16 INK4A (#92803), p21 Cip1 (#2947), phospho(T202/Y204)-Erk1/2 (#9106), Erk1/2 (#9107), phospho(S139)-H2A.X (#9718), phospho(S15)-p53 (#92845), phospho(S46)-p53 (#2521), phospho(S780)-Rb (#9307), β-actin (#4970); caspase-3 (#9665), cleaved caspase-3 (#9664), PTMScan ® O-GlcNAc [GlcNAc-S/T] Motif Kit (#95220).

Techniques: Modification

Journal: bioRxiv

Article Title: Mapping the endothelial O-GlcNAcome uncovers CCAR1 as a regulator of senescence

doi: 10.64898/2026.02.12.705616

Figure Lengend Snippet: (A) Lysates of control or Thiamet G-treated HUVEC were immunoprecipitated for CCAR1 and stained by immunoblot for O-GlcNAc (upper panel) and CCAR1 (lower panel). Images are representative of 3 independent experiments using cells from different donors. (B) MS-based analysis of enriched proteins after CCAR1 co-immunoprecipitation. Coverage of the N-terminal region of CCAR1 by LC-MS using chymotrypsin as protease. HexNAc modified peptides are indicated in red. (C) MS-intensity fold change between Thiamet G-treatment and control, normalized to protein fold change, for the two most abundant HexNAc peptides and their unmodified counterpart. Data are means ± SD of 3 independent experiments using cells from different donors. (D, E) Representative MS/MS spectrum of the HexNAc modified peptide “TATAVSQPAALGVQQPSLL” (D) and “TQPAVALPTSL” (E) . Peak colors: orange, green and blue refer to y, b and diagnostic oxonium ions, respectively. The enlarged view in the m/z range of 100 to 225 (D) shows the [HexNAc]+ ion (C8H14NO5) and corresponding fragmentation induced oxonium ions.

Article Snippet: The following antibodies were purchased from Cell Signalling Technology: O-GlcNAc (CTD110.6 clone, #9875), OGT (#24083), p16 INK4A (#92803), p21 Cip1 (#2947), phospho(T202/Y204)-Erk1/2 (#9106), Erk1/2 (#9107), phospho(S139)-H2A.X (#9718), phospho(S15)-p53 (#92845), phospho(S46)-p53 (#2521), phospho(S780)-Rb (#9307), β-actin (#4970); caspase-3 (#9665), cleaved caspase-3 (#9664), PTMScan ® O-GlcNAc [GlcNAc-S/T] Motif Kit (#95220).

Techniques: Control, Immunoprecipitation, Staining, Western Blot, Liquid Chromatography with Mass Spectroscopy, Modification, Tandem Mass Spectroscopy, Diagnostic Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Repeated hypoxia exposure induces cognitive dysfunction, brain inflammation, and amyloidβ/p-Tau accumulation through reduced brain O-GlcNAcylation in zebrafish

doi: 10.1177/0271678x211027381

Figure Lengend Snippet: Figure 4. Effects of GlcN on OGA and OGT expression and PKA/CREB signaling in control, SH and RH zebrafish. Zebrafish were exposed to SH (1) or RH (5) with or without intraperitoneal pre-injection of GlcN (G, 200 lg/g) at 2 h prior to each bout of hypoxia. Representative confocal images (x40) of DAPI (blue), OGT (green) and OGA (red, A), and p-CREB (green,B) and PKAca (red, B) and merged immunofluorescence staining of the Dl of zebrafish brain at 6 h after FC in the control (normoxia, CON or C), SH (1), and RH (5) hypoxic (HYP or H) groups. Enlarged images are presented in the white boxes. Graphs represent densito- metric quantification of OGT, OGA, p-CREB or PKAca (n¼3–5/group). Statistical analysis was carried out by Kruskal-Wallis with false discovery rate (FDR) multiple comparison test. (*p<0.05, **p<0.01 vs. Cont, #p<0.05, ##p<0.01 vs. fear-conditioned Cont, §p<0.05, §§p<0.01 vs. fear-conditioned RH.)

Article Snippet: For staining, the slides were incubated with heated citrate buffer (10mM citric buffer, pH 6.0) for 5min, rinsed in 0.1% Triton X-100 in PBS (PBS-T), and blocked with 10% normal goat serum (Jackson ImmunoResearch, West Grove, PA) in PBS-T. After being washed, the sections were incubated in PBS-T solutions containing antibodies specific for OGlcNAc, phospho-CREB, OGT, GFAP, p65, p50 and c-Rel (Santa Cruz Biotechnology, Dallas, TX), OGA (Proteintech, Rosemont, IL), PKA-ca, b-amyloid (Cell signaling Technology, Danvers, MA), L-plastin, p-Tau (Abcam, Cambridge, UK) or S100b (DAKO, Glostrup, Denmark) at 4 C. The sections were then rinsed with 0.1% PBS-T and incubated with a mixture of secondary antibodies, including goat anti-mouse or rabbit IgG conjugated to Alexa Flour 488 or 568 (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Control, Injection, Immunofluorescence, Staining, Comparison

Journal: PLoS ONE

Article Title: Identification of Nitric Oxide as an Endogenous Inhibitor of 26S Proteasomes in Vascular Endothelial Cells

doi: 10.1371/journal.pone.0098486

Figure Lengend Snippet: OGT siRNA knockdown abolished the proteasome reporter protein accumulation and Rpt2 O-GlcNAcylation induced by (A) SNP; (B) Bradykinin; and (C) A23187. Rpt2 O-GlcNAcylation was detected with the WGA protocol. The shown blots were representative of at least 3 independent experiments with similar results. OGT knockdown by siRNA restored 26S proteasome activity in cell treated with (D) SNP; (E) Bradykinin; and (F) A23187. 26S proteasome activity was quantified by measuring chymotrypsin-like activity in the cell lysates. * represent p<0.05 vs control (n = 3). An overlaid portion (less exposure) of each whole blot indicating Ub-GFP is presented. Ctrl, control (scrambled) siRNA; NS, not significant (v.s. control); OGT, O-GlcNAc transferase; SNP, sodium nitroprusside; Ub-GFP, ubiquitin-green fluorescent protein; WGA, wheat germ agglutinin.

Article Snippet: Transfection of control or target (OGT) siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA) as described previously .

Techniques: Knockdown, Activity Assay, Control, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Identification of Nitric Oxide as an Endogenous Inhibitor of 26S Proteasomes in Vascular Endothelial Cells

doi: 10.1371/journal.pone.0098486

Figure Lengend Snippet: (A) Ub G76V -GFP-expressing HUVEC were transfected either with control or OGA siRNA and cell lysates were subjected to Western blot to detect reporter protein levels and Rpt2 O-GlcNAcylation. (B) chymotrypsin-like activity in siRNA treated cells. (C) Adenoviral overexpression of OGA, not GFP, decreased both the levels of reporter protein and Rpt2 O-GlcNAcylation. (D) chymotrypsin-like activity in adenovirus infected cells. The blots shown were representative of 3 independent experiments with similar results. * represents p<0.05 vs the control (n = 3). An overlaid portion (less exposure) of each whole blot indicating Ub-GFP is presented. Ctrl, control (scrambled) siRNA; OGA, O-GlcNAcase; OGT, O-GlcNAc transferase; Ub-GFP, ubiquitin-green fluorescent protein; WGA, wheat germ agglutinin.

Article Snippet: Transfection of control or target (OGT) siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA) as described previously .

Techniques: Expressing, Transfection, Control, Western Blot, Activity Assay, Over Expression, Infection, Ubiquitin Proteomics

Journal: Aging (Albany NY)

Article Title: Sevoflurane postconditioning reduces myocardial ischemia reperfusion injury-induced necroptosis by up-regulation of OGT-mediated O-GlcNAcylated RIPK3

doi: 10.18632/aging.104146

Figure Lengend Snippet: SPC up-regulated the expression of O-GlcNAc and OGT proteins. ( A ) Representative protein images of O-GlcNAc, OGT and OGA from all groups were shown. ( B ) Quantitative analysis of O-GlcNAc, OGT and OGA proteins were performed. n = 3 /group. The columns and errors bars represent means ± SD. * P < 0.05 vs. SHAM group; # P < 0.05 vs. IR group.

Article Snippet: After blocking with 5% non-fat milk at room temperature for 1 h, primary antibodies against p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, MLKL (1:1000 dilution, Thermo Fisher Co., Massachusetts, USA), O-GlcNAc, OGT, OGA (1:1000 dilution, Santa Cruz Co., California, USA) were incubated with the PVDF membranes at 4° C overnight.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Sevoflurane postconditioning reduces myocardial ischemia reperfusion injury-induced necroptosis by up-regulation of OGT-mediated O-GlcNAcylated RIPK3

doi: 10.18632/aging.104146

Figure Lengend Snippet: SPC increased O-GlcNAcylated RIPK3 and inhibited the combination of RIPK3 and MLKL. ( A ) RIPK3 and MLKL were detected in O-GlcNAc immunoprecipitates. Representative protein images were performed. ( B ) Quantitative analysis of O-GlcNAcylated RIPK3 and MLKL. n = 3 /group. ( C ) O-GlcNAc and MLKL in RIPK3 immunoprecipitates. Representative protein images were shown. ( D ) Quantitative analysis of O-GlcNAcylated RIPK3 and the complex between RIPK3 with MLKL. n = 3 /group.

Article Snippet: After blocking with 5% non-fat milk at room temperature for 1 h, primary antibodies against p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, MLKL (1:1000 dilution, Thermo Fisher Co., Massachusetts, USA), O-GlcNAc, OGT, OGA (1:1000 dilution, Santa Cruz Co., California, USA) were incubated with the PVDF membranes at 4° C overnight.

Techniques:

Journal: Aging (Albany NY)

Article Title: Sevoflurane postconditioning reduces myocardial ischemia reperfusion injury-induced necroptosis by up-regulation of OGT-mediated O-GlcNAcylated RIPK3

doi: 10.18632/aging.104146

Figure Lengend Snippet: OSMI-1 abolished SPC induced activation in OGT mediated O-GlcNAcylation and reduction in p-RIPK3, p-MLKL. ( A , B ) Expressions of O-GlcNAc, OGT and OGA in all groups were analyzed by Western Blot. Representative protein images and quantitative analysis were shown. n=3/group. ( C , D ) Representative protein images and quantitative analysis of p-RIPK1, p-RIPK3, and p-MLKL in all groups were presented. n=3/group. OSMI-1, the OGT inhibitor; DMSO, the OSMI-1 solvent. The columns and errors bars represent means ± SD. * P < 0.05 vs. SHAM group; # P < 0.05 vs. IR group; & P < 0.05 vs. SPC group.

Article Snippet: After blocking with 5% non-fat milk at room temperature for 1 h, primary antibodies against p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, MLKL (1:1000 dilution, Thermo Fisher Co., Massachusetts, USA), O-GlcNAc, OGT, OGA (1:1000 dilution, Santa Cruz Co., California, USA) were incubated with the PVDF membranes at 4° C overnight.

Techniques: Activation Assay, Western Blot, Solvent

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Best practices in assessing cardiac protein O -GlcNAcylation by immunoblot

doi: 10.1152/ajpheart.00104.2023

Figure Lengend Snippet: List of all antibodies used in this study including vendors, catalog numbers, dilutions, and concentrations

Article Snippet: HGAC85 , Novus Biologicals , NB300-614 , 1:1,000 , N/A.

Techniques: Concentration Assay

Journal: Current Protocols in Molecular Biology

Article Title: Detection and Analysis of Proteins Modified by O-Linked N-Acetylglucosamine

doi: 10.1002/0471142727.mb1706s95

Figure Lengend Snippet: Antbodies and Lectins that Recognize O-GlcNAc and GlcNAc

Article Snippet: BSA-AP- GlcNAc BSA-AP or Ovalbumin ( Matsuoka et al. 2002 ) 18B10.C7(3) IgG O-GlcNAc Millipore BSA-AP- GlcNAc BSA-AP or Ovalbumin ( Teo et al. 2010 ) 9D1.E4(10) IgG O-GlcNAc Millipore BSA-AP- GlcNAc BSA-AP or Ovalbumin ( Teo et al. 2010 ) 1F5.D6(14) IgG O-GlcNAc Millipore BSA-AP- GlcNAc BSA-AP or Ovalbumin ( Teo et al. 2010 ) 6D93 IgG O-GlcNAc Santa Cruz Biotechnology BSA-AP- GlcNAc BSA-AP or Ovalbumin Santa Cruz 10D8 IgM O-GlcNAc Santa Cruz Biotechnology BSA-AP- GlcNAc BSA-AP or Ovalbumin Santa Cruz HGAC 85 IgG GlcNAc Novus Biologicals, Abcam, Pierce, Enzo life sciences, Affinity Bioreagents BSA-AP- GlcNAc, Ovalbumin BSA-AP or BSA ( Turner et al. 1990 ) HGAC 49 IgM GlcNAc No BSA-AP- GlcNAc, Ovalbumin BSA-AP or BSA ( Turner et al. 1990 ) HGAC 39 IgG GlcNAc No BSA-AP- GlcNAc, Ovalbumin BSA-AP or BSA ( Turner et al. 1990 ) Open in a separate window † BSA-AP, BSA-aminophenol * succinylation of WGA (sWGA) reduces it’s affinity for Sialic Acid.

Techniques: Positive Control, Negative Control