rabbit polyclonal anti p enos ser1177  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: Regulation of O-GlcNAcylation on endothelial nitric oxide synthase by glucose deprivation and identification of its O-GlcNAcylation sites

doi: 10.1038/s41598-020-76340-7

Figure Lengend Snippet: Effect of glucose deprivation on bovine aortic endothelial cell (BAEC) endothelial nitric oxide synthase (eNOS) O-GlcNAcylation. ( A ) Left panel: representative western blot of BAEC eNOS O-GlcNAcylation and phospho-eNOS (Ser1177) from control or glucose deprivation-treated cells (n = 3) over 10 h. Right panel: quantification of the ratio between O-GlcNAcylation, phosphorylated eNOS (P-eNOS), and eNOS. ( B ) Left panel: representative western blot of BAEC O-GlcNAcylation, P-eNOS, and eNOS in response to a decrease in extracellular glucose from 5.5 to 0 mM (n = 3) over 6 h. Right panel: quantification of the ratio between O-GlcNAcylation, P-eNOS, and eNOS. ( C ) Left panel: representative western blot of BAEC eNOS O-GlcNAcylation and phospho-eNOS (Ser1177) from control or 1 mM glucose-treated cells (n = 3) over 10 h. Right panel: quantification of the ratio between O-GlcNAcylation, P-eNOS, and eNOS. Full-length blots are presented in Supplementary Figure (A), (B), and (C). Data are shown as the mean ± SEM. Signal density of O-GlcNAc and P-eNOS (Ser-1177) bands relative to the corresponding eNOS bands. Values are normalized to control (0 h). * P < 0.05 vs. control. Squares = O-GlcNAc; circles = P-eNOS (Ser 1179).

Article Snippet: Membranes used for O-GlcNAc analysis were stripped and probed with rabbit polyclonal anti-P-eNOS (Ser-1177) Ab (Cell Signaling Technology), and then stripped and reprobed for total eNOS.

Techniques: Western Blot, Control

Journal: Scientific Reports

Article Title: Regulation of O-GlcNAcylation on endothelial nitric oxide synthase by glucose deprivation and identification of its O-GlcNAcylation sites

doi: 10.1038/s41598-020-76340-7

Figure Lengend Snippet: Increased O-GlcNAcylation stimulated by glucose deprivation is caused by AMP kinase (AMPK)-dependent O-GlcNAc transferase (OGT) activation. ( A ) Left panel: representative western blot of OGT and O-GlcNAcase (OGA) from control (5.5 mM) or low (1 mM) glucose (LG)-treated bovine aortic endothelial cells (BAECs) (n = 3) incubated for 6 h. Right panel: quantification of the ratio between OGT and OGA. Signal density of OGT and OGA bands relative to the corresponding eNOS bands. ( B ) Left panel: representative western blot of phospho-threonine OGT and OGT from control or LG-treated BAECs (n = 3) incubated for 6 h. Right panel: quantification of the ratio between phospho-threonine OGT and OGT. ( C ) Left panel: representative western blot of AMPK, P-AMPK, eNOS O-GlcNAcylation, and eNOS from control or LG-treated BAECs or AMPK-knockdown BAECs (n = 3) incubated for 6 h. Right panel: quantification of the ratio of AMPK, P-AMPK, eNOS O-GlcNAcylation, and eNOS. Signal density of O-GlcNAcylation and OGT bands relative to the corresponding eNOS bands. Signal density of AMPK bands relative to the corresponding β-actin bands. Signal density of p-AMPK bands relative to the corresponding AMPK bands. AICAR was an activator of AMPK. Full-length blots are presented in Supplementary Figure (A), (B), and (C). Data are shown as the mean ± SEM, * < 0.05; ** P < 0.01 vs. control.

Article Snippet: Membranes used for O-GlcNAc analysis were stripped and probed with rabbit polyclonal anti-P-eNOS (Ser-1177) Ab (Cell Signaling Technology), and then stripped and reprobed for total eNOS.

Techniques: Activation Assay, Western Blot, Control, Incubation, Knockdown

Journal: Scientific Reports

Article Title: Regulation of O-GlcNAcylation on endothelial nitric oxide synthase by glucose deprivation and identification of its O-GlcNAcylation sites

doi: 10.1038/s41598-020-76340-7

Figure Lengend Snippet: Effect of hypoglycemia on BAEC eNOS O-GlcNAcylation in rat aortae. ( A ) Blood glucose levels in response to insulin-induced hypoglycemia (200 g/U, subcutaneous) in a rat model (n = 5). ( B ) Left panel: representative western blot of O-linked Glc-NAc, P-eNOS, and eNOS from the thoracic aorta of control or insulin-treated rats injected subcutaneously with insulin (n = 5) at 3–9 h after injection. Right panel: quantification of the ratio between O-GlcNAcylation, P-eNOS, and eNOS. Data are shown as the mean ± SEM and signal density of O-GlcNAc and P-eNOS (Ser-1177) bands relative to the corresponding total eNOS bands. Full-length blots are presented in Supplementary Figure . ** P < 0.01 vs. Control (0 h).

Article Snippet: Membranes used for O-GlcNAc analysis were stripped and probed with rabbit polyclonal anti-P-eNOS (Ser-1177) Ab (Cell Signaling Technology), and then stripped and reprobed for total eNOS.

Techniques: Western Blot, Control, Injection

Journal: Scientific Reports

Article Title: Regulation of O-GlcNAcylation on endothelial nitric oxide synthase by glucose deprivation and identification of its O-GlcNAcylation sites

doi: 10.1038/s41598-020-76340-7

Figure Lengend Snippet: Effect of glucose deprivation on O-GlcNAcylation and phospho-eNOS (Ser1177) in His-tagged wild-type (WT)-eNOS and S1177A-eNOS. ( A ) Left panel: representative western blot of His-tagged WT-eNOS-transfected HEK293 cells, O-GlcNAcylation and phospho-eNOS (Ser1177) from control or low glucose-treated cells (n = 3) incubated for 6 h. Right panel: quantification of the ratio of O-GlcNAcylation and phospho-eNOS (Ser1177). ( B ) Left panel: representative western blot of WT-eNOS- or S1177A-eNOS-transfected HEK293 cells showing O-GlcNAcylation, phospho-eNOS (Ser1177), phospho-eNOS (Thr495), and phospho-eNOS (Ser633) under normal culture conditions. Right panel: quantification of the ratio of O-linked GlcNAc, phospho-eNOS (Ser1177), phospho-eNOS (Thr495), and phospho-eNOS (Ser633). White = WT-eNOS; black = S1177A-eNOS ( C ) Left panel: representative western blot of S1177A-eNOS-transfected HEK293 cells showing O-GlcNAcylation and phospho-eNOS (Ser1177) in control or low glucose-treated cells (n = 3) incubated for 6 h. Right panel: quantification of the ratio of O-GlcNAcylation and phospho-eNOS (Ser1177). ( D ) Left panel: representative western blot of WT-eNOS- and S1177A-eNOS-transfected BAECs showing O-GlcNAcylation and phospho-eNOS (Ser1177) in control or low glucose-treated cells (n = 3) incubated for 6 h. Right panel quantification of the ratio of O-GlcNAcylation and phospho-eNOS (Ser1177). Data are shown as the mean ± SEM and signal density of O-GlcNAc and P-eNOS bands relative to the corresponding total eNOS bands. Control, 5 Mm glucose; LG, 1 mM glucose. Full-length blots are presented in Supplementary Figure (A), (B), (C) and (D). * P < 0.05, ** P < 0.01 vs. control.

Article Snippet: Membranes used for O-GlcNAc analysis were stripped and probed with rabbit polyclonal anti-P-eNOS (Ser-1177) Ab (Cell Signaling Technology), and then stripped and reprobed for total eNOS.

Techniques: Western Blot, Transfection, Control, Incubation

Journal: Scientific Reports

Article Title: Regulation of O-GlcNAcylation on endothelial nitric oxide synthase by glucose deprivation and identification of its O-GlcNAcylation sites

doi: 10.1038/s41598-020-76340-7

Figure Lengend Snippet: High-pressure liquid chromatography-mass spectrometry (HPLC–MS) and MS/MS Ion Search for enriched eNOS O-GlcNAcylation sites. ( A ) Inset, eNOS protein was recovered from gel. Fractionation by SDS-PAGE revealed a single major eNOS band, which was analyzed by HPLC–MS. ( B ) Left panel: MS/MS Ion Search score of eNOS Ser867 O-GlcNAcylation site. Upper right panel: representative western blot of S867A-eNOS-transfected HEK293 cells showing O-linked GlcNAc from control or low glucose -treated cells (n = 3) incubated for 6 h. Lower right panel: quantification of the ratio between control and low glucose treated O-GlcNAcylation. ( C ) Left panel: MS/MS Ion Search score of eNOS Thr866 O-GlcNAcylation site. Upper right panel: representative western blot of T866A-eNOS-transfected HEK293 cells showing O-linked GlcNAc from control or low glucose -treated cells (n = 3) incubated for 6 h. Lower right panel: quantification of the ratio between control and low glucose treated O-GlcNAcylation. ( D ) Left panel: representative western blot of T866A-eNOS-transfected BAECs showing O-GlcNAcylation from control or low glucose -treated cells (n = 3) incubated for 6 h. Right panel: quantification of the ratio between control and low glucose treated O-GlcNAcylation. Data are shown as the mean ± SEM and signal density of O-GlcNAc and P-eNOS bands relative to the corresponding total eNOS bands. Control, 5 Mm glucose; LG, 1 mM glucose. Full-length blots are presented in Supplementary Figure (A), (B), (C) and (D). ** P < 0.01 vs. control.

Article Snippet: Membranes used for O-GlcNAc analysis were stripped and probed with rabbit polyclonal anti-P-eNOS (Ser-1177) Ab (Cell Signaling Technology), and then stripped and reprobed for total eNOS.

Techniques: High Performance Liquid Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy, Fractionation, SDS Page, Western Blot, Transfection, Control, Incubation

Journal: Scientific Reports

Article Title: Regulation of O-GlcNAcylation on endothelial nitric oxide synthase by glucose deprivation and identification of its O-GlcNAcylation sites

doi: 10.1038/s41598-020-76340-7

Figure Lengend Snippet: Effect of glucose deprivation on eNOS activity. ( A ) NO release in BAEC culture medium in control or low glucose-treated BAECs (n = 3) over 10 h. White = control; black = low glucose (LG). ( B ) NO release in rat aorta plasma from control or low glucose-treated rat (n = 3) after 6 h. ( C ) Cumulative concentration–response curves to acetylcholine (10 −8 –10 −4.5 M) in aortic segments of hypoglycemia or AMPK inhibitor-treated hypoglycemia rat. Compound C (0.2 mg/kg, i.v.). Compound C was administrated 30 min before insulin (200 g/U) injection. Ach, Acetylcholine. ( D ) Effect of low glucose on L-[14C] citrulline in WT-eNOS- and T866A-eNOS-transfected HEK293 cells. Data are shown as the mean ± SEM and Control, 5 Mm glucose; LG, 1 mM glucose. Full-length blots are presented in Supplementary Figure . * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control; $ represents hypoglycemia vs. hypoglycemia + Dorsomorphin, P < 0.05.

Article Snippet: Membranes used for O-GlcNAc analysis were stripped and probed with rabbit polyclonal anti-P-eNOS (Ser-1177) Ab (Cell Signaling Technology), and then stripped and reprobed for total eNOS.

Techniques: Activity Assay, Control, Clinical Proteomics, Concentration Assay, Injection, Transfection