Journal: Chinese Medicine

Article Title: Cinnamic acid alleviates endothelial dysfunction and oxidative stress by targeting PPARδ in obesity and diabetes

doi: 10.1186/s13020-025-01064-7

Figure Lengend Snippet: The impact of cinnamic acid on endothelial nitric oxide synthase (eNOS) and antioxidant pathways in mouse aortas. Representative bands and densitometry of the protein levels of a PPARδ (52 kDa), b Nrf2 (110 kDa), and c HO-1 (33 kDa) relative to GAPDH (36 kDa), d phosphorylated (p)-AMPKα (at Thr172) relative to total AMPKα (62 kDa), e p-Akt (at Ser473) relative to total Akt (60 kDa), and f p-eNOS (at Ser1177) relative to total eNOS (140 kDa). # P < 0.05 vs. control (CON) group. * P < 0.05 vs. diet-induced obese (DIO) group. Data were shown as mean ± SEM (n = 6–8)

Article Snippet: Antibodies against phosphorylated AMP-activated protein kinase α (AMPKα) (Thr172), total AMPKα, phosphorylated eNOS (Ser1177) were obtained from Proteintech Group (USA).

Techniques: Control

Journal: Chinese Medicine

Article Title: Cinnamic acid alleviates endothelial dysfunction and oxidative stress by targeting PPARδ in obesity and diabetes

doi: 10.1186/s13020-025-01064-7

Figure Lengend Snippet: The impact of cinnamic acid on endothelial nitric oxide synthase (eNOS) and antioxidant pathways in rat aortic endothelial cells (RAECs). Representative bands and densitometry of the protein levels of a PPARδ (52 kDa), b Nrf2 (110 kDa), and c HO-1 (33 kDa) relative to GAPDH (36 kDa), d phosphorylated (p)-AMPKα (at Thr172) relative to total AMPKα (62 kDa), e p-Akt (at Ser473) relative to total Akt (60 kDa), and f p-eNOS (at Ser1177) relative to total eNOS (140 kDa) in high glucose (HG, 44.4 mM)-induced RAECs. # P < 0.05 vs. normal glucose (NG) group. * P < 0.05 vs. HG group. Data were shown as mean ± SEM (n = 4–6)

Article Snippet: Antibodies against phosphorylated AMP-activated protein kinase α (AMPKα) (Thr172), total AMPKα, phosphorylated eNOS (Ser1177) were obtained from Proteintech Group (USA).

Techniques:

Journal: Journal of the American Heart Association

Article Title: Interleukin‐1β Disruption Protects Male Mice From Heart Failure With Preserved Ejection Fraction Pathogenesis

doi: 10.1161/jaha.122.029668

Figure Lengend Snippet: Figure 3. Western blot analysis and nitrate/nitrite levels. Western blot analysis and cumulative data for phosphorylated endothelial nitric-oxide synthase (P-eNOS) in mesenteric resistance arteries (MRAs) isolated from C57/BL6J (C57), C57/BL6J+high-fat diet (HFD)+N[w]-nitro-l-arginine methyl ester (L-NAME), interleukin- 1β−/− (IL-1β−/−), and IL-1β−/−+HFD+L-NAME male mice (n=5) (A) and nitrate/nitrite level in the urine (B) from all groups of mice. One-way ANOVA followed by Tukey’s multiple comparisons post hoc test was applied for (A and B). *P<0.05, ****P<0.0001 for C57BL/6J vs C57/ Bl6+HFD+L-NAME; (n=5). ns indicates not significant; and T-eNOS, total nitric oxide synthase.

Article Snippet: We used specific antibodies against endothelial nitric oxide synthase (eNOS; Cell Signaling, D9A5L rabbit monoclonal antibodies; number 32027), phospho- eNOS (Ser1177; Cell D ow nloaded from http://ahajournals.org by on January 26, 2024 Signaling [C9C3] rabbit monoclonal antibodies, 9570), iNOS (Proteintech, number 18985- 1- AP), anti- COX2 (Cayman; aa 584– 598, number 160126), NLR family pyrin domain containing 3/Nacht leucine- rich repeat protein 3 (Novus Biotech; number NBP2- 12446), X- box binding protein 1 (Abcam; number ab238456), anti– CCAAT/enhancer- binding protein (Cell Signaling; L63F7 mouse monoclonal antibodies number 2895), IRE1 (Abcam; number ab37073, lot GR3354835- 4), activating transcription factor 6 (Abcam; number ab37149, lot GR3353621- 3), and beta- actin (Santa Cruz; number SC47778).

Techniques: Western Blot, Isolation

Journal: Scientific Reports

Article Title: Activated ROCK/Akt/eNOS and ET-1/ERK pathways in 5-fluorouracil-induced cardiotoxicity: modulation by simvastatin

doi: 10.1038/s41598-020-71531-8

Figure Lengend Snippet: Effect of 5-FU and Sim treatments on cardiac tissue contents of ( A ) p -Akt and ( B ) p -eNOS. Values are presented as the mean of 6–7 experiments ± SEM. Statistical analysis was carried out using one-way ANOVA followed by Tukey’s post-hoc test ; as compared to normal (*), Sim (#), and 5-FU (@)-treated groups (p < 0.05). 5-FU 5-fluorouracil, p-Akt (Ser473) phosphorylated protein kinase B at Ser 473, p-eNOS (Ser1177) phosphorylated endothelial nitric oxide synthase at Ser 1177, p-ERK1/2 (Thr202/Tyr204) phosphorylated extracellular signal-regulated kinase 1/2 at Thr 202 & Tyr 204, Sim simvastatin.

Article Snippet: The corresponding ELISA kits were used to determine the cardiac contents of p -NF-κB p65 (Ser536) (Abcam, Cambridge, UK; Cat# ab176647), p -ERK1/2 (Thr202/Tyr204) (Invitrogen, CA, USA; Cat # EMS2ERKP), p -Akt (Ser473) (Elabscience, Wuhan, China; Cat # E-EL-R1135) and p -eNOS (Ser1177) (Cell Signaling Technology, MA, USA; Cat# 7980C).

Techniques:

Journal: Experimental Physiology

Article Title: Acute hypobaric hypoxia and cardiac energetic response in prepubertal rats: Role of nitric oxide

doi: 10.1113/ep089064

Figure Lengend Snippet: FIGURE 4 (a) Left ventricular cytosolic p-eNOS-Ser1177 expression. (b) Left ventricular cytosolic endothelial nitric oxide synthase (eNOS) expression. (c) Left ventricular cytosolic inducible nitric oxide synthase (iNOS) expression. (d) Left ventricular cytosolic neuronal nitric oxide synthase (nNOS) expression. Abbreviations: HH, acute hypobaric hypoxia group; N, normoxic group. Values are means ± SD. *P < 0.05 versus N group

Article Snippet: Blots were probed with a 1:1000 dilution of primary antibody specific for p-eNOS (rabbit monoclonal, #9570; Cell Signaling Technology, Danvers, MA, USA), eNOS (rabbit polyclonal, #sc-654; Santa Cruz Biotechnology, Santa Cruz, CA, USA), nNOS (rabbit, amino terminus, H-299; Santa Cruz Biotechnology), iNOS (mouse monoclonal, #sc-7271; Santa Cruz Biotechnology) or βtubulin (mouse monoclonal, #ab131205; Abcam, Cambridge, United Kingdom).

Techniques: Expressing

Journal: Experimental Physiology

Article Title: Acute hypobaric hypoxia and cardiac energetic response in prepubertal rats: Role of nitric oxide

doi: 10.1113/ep089064

Figure Lengend Snippet: FIGURE 6 Hypothetical main myocyte differential molecular response activated during acute hypobaric hypoxia (HH) related to age. (a) In prepubertal rats, HH increased nitric oxide (NO) production by vascular endothelial cells through their own endothelial NOS (eNOS), triggering, by negative feedback over the nucleus of the surrounding myocytes, a reduction of mRNA and nitric oxide synthase (NOS) synthesis (dashed boxes). That reduction in NO production by NOS might be compensated by xanthine oxidoreductase (XOR), which would produce NO from the nitrite–nitrate (Nx) reservoir, coming largely from the diet (Moretti et al., 2019) and possibly also from the previously oxidized vascular NO mentioned above. This maintains normal intracellular NO levels, resulting in a maintenance of mitochondrial and contractile function [left ventricular develop pressure (LVDP)] as in normoxic conditions, preventing the development of endogenous cardioprotection (LVDP dashed box). The increased heat (Ht) detected could be attributed to a major relative participation of active transport mechanisms, such as the Na+–Ca2+

Article Snippet: Blots were probed with a 1:1000 dilution of primary antibody specific for p-eNOS (rabbit monoclonal, #9570; Cell Signaling Technology, Danvers, MA, USA), eNOS (rabbit polyclonal, #sc-654; Santa Cruz Biotechnology, Santa Cruz, CA, USA), nNOS (rabbit, amino terminus, H-299; Santa Cruz Biotechnology), iNOS (mouse monoclonal, #sc-7271; Santa Cruz Biotechnology) or βtubulin (mouse monoclonal, #ab131205; Abcam, Cambridge, United Kingdom).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Thromboxane A2 Receptor Activates a Rho-associated Kinase/LKB1/PTEN Pathway to Attenuate Endothelium Insulin Signaling

doi: 10.1074/jbc.m109.012583

Figure Lengend Snippet: FIGURE 1. TP activation impairs both basal and insulin-stimulated eNOS-Ser1177 phosphorylation in HUVECs. A, TP activation impairs basal eNOS phosphorylation. Confluent HUVECs cultured in 0.5% FBS were pretreated with TP antagonist (SQ29548, 4 mol/liter) for 20 min and exposed to TP agonist (IBOP or U46619, 0.4 mol/liter) overnight. The cells were then harvested for detection of eNOS phosphorylation (n 5; *, p 0.05 versus control (Ctrl); †, p 0.05 versus IBOP or U46619). B, TP activation impairs insulin-stimulated eNOS phosphorylation. HUVECs were treated as described above and then stimulated with insulin for 10 min. The cells were then harvested for analysis of eNOS-Ser1177 phosphorylation (n 5; *, p 0.05 versus insulin; †, p 0.05 versus IBOP insulin or U46619 insulin). C, TP is required for the IBOP action. HUVECs were transfected with either control siRNA or TP siRNA (100 nmol/liter) for 48 h and treated with IBOP (0.4 mol/liter, overnight). TP siRNA significantly knocked down both TP and TP isoforms in HUVECs as immunoblotted with antibody from Cayman Chemical. The blot is representative of three blots obtained from three separate experiments (n 3; *, p 0.05 versus control; †, p 0.05 versus control siRNA IBOP). D, TP is required for the inhibition of insulin signaling by IBOP. The siRNA-transfected HUVECs were treated with IBOP (0.4 mol/liter, overnight) and then stimulated with insulin (n 3; *, p 0.05 versus control siRNA insulin; †, p 0.05 versus control siRNA IBOP insulin).

Article Snippet: Recombinant human ROCK1 and ROCK2 proteins and antibodies against PTEN, phosphoPTEN-Ser380/Thr382/383, Akt, phospho-Akt-Ser473, eNOS, phospho-eNOS-Ser1177, phosphoLKB1-Ser428, and -tubulin were obtained from Cell Signaling Technology (Beverly,MA).

Techniques: Activation Assay, Phospho-proteomics, Cell Culture, Control, Transfection, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Thromboxane A2 Receptor Activates a Rho-associated Kinase/LKB1/PTEN Pathway to Attenuate Endothelium Insulin Signaling

doi: 10.1074/jbc.m109.012583

Figure Lengend Snippet: FIGURE 3. TP activation impairs both basal and insulin-stimulated eNOS-Ser1177 and Akt-Ser473 phos- phorylation in MAECs. A and B, TP activation impairs basal eNOS and Akt phosphorylation. Confluent MAECs cultured in 0.5% FBS were pretreated with TP antagonist (SQ29548, 4 mol/liter) for 20 min and exposed to TP agonist (IBOP or U46619, 0.4 mol/liter) overnight. The cells were then harvested for detection of (A) eNOS and (B) Akt phosphorylation (n 3; *, p 0.05 versus control; †, p 0.05 versus IBOP or U46619). C and D, TP activation impairs insulin-stimulated eNOS and Akt phosphorylation. MAECs were treated as described above and then stimulated with insulin for 10 min. The cells were then harvested for the analysis of eNOS and Akt phosphorylation (n 3; *, p 0.05 versus insulin; †, p 0.05 versus IBOP insulin or U46619 insulin). E and F, TP activation impairs both basal and insulin-stimulated phosphorylation of Akt-Ser473 in human aortic endo- thelial cells. Confluent human aortic endothelial cells cultured in 0.5% FBS were pretreated with TP antagonist (SQ29548, 4 mol/liter) for 20 min and exposed to TP agonists (IBOP or U46619, 0.4 mol/liter) overnight (E) or then treated with insulin (F). The cells were then harvested for the detection of Akt phosphorylation. The blot is a representative of two blots obtained from two independent experiments.

Article Snippet: Recombinant human ROCK1 and ROCK2 proteins and antibodies against PTEN, phosphoPTEN-Ser380/Thr382/383, Akt, phospho-Akt-Ser473, eNOS, phospho-eNOS-Ser1177, phosphoLKB1-Ser428, and -tubulin were obtained from Cell Signaling Technology (Beverly,MA).

Techniques: Activation Assay, Phospho-proteomics, Cell Culture, Control

Journal: Journal of Biological Chemistry

Article Title: Thromboxane A2 Receptor Activates a Rho-associated Kinase/LKB1/PTEN Pathway to Attenuate Endothelium Insulin Signaling

doi: 10.1074/jbc.m109.012583

Figure Lengend Snippet: FIGURE 9. TP mediates HFD-induced inhibition of both Akt and eNOS in murine aortas. Male C57BL/6J mice were fed a normal diet (ND) or a HFD for 13 weeks and administrated vehicle or SQ29548 (5 g/kg/day) for the following 5 weeks. A–C, aortic homogenates were analyzed for levels of total and phosphorylated eNOS (phosphorylation at Ser1177) (A), Akt (phosphorylation at Ser473) (B), and PTEN (phosphorylation at Ser380/ Thr382/383) (C). D, proposed pathway underlying TP-induced attenuation of insulin signaling.

Article Snippet: Recombinant human ROCK1 and ROCK2 proteins and antibodies against PTEN, phosphoPTEN-Ser380/Thr382/383, Akt, phospho-Akt-Ser473, eNOS, phospho-eNOS-Ser1177, phosphoLKB1-Ser428, and -tubulin were obtained from Cell Signaling Technology (Beverly,MA).

Techniques: Inhibition, Phospho-proteomics

Journal: Hypertension research : official journal of the Japanese Society of Hypertension

Article Title: Preventive effect of gomisin J from Schisandra chinensis on angiotensin II-induced hypertension via an increased nitric oxide bioavailability.

doi: 10.1038/hr.2014.162

Figure Lengend Snippet: Figure 3 Effect of gomisin J (GJ) on impaired nitric oxide (NO) production in endothelial cells treated with angiotensin II (Ang II). (a) Representative photographs of the preservation of NO production by GJ in endothelial cells treated with Ang II (10 μM) (n = 5). (b and c) Representative immunoblots showing the effect of GJ on endothelial nitric oxide synthase (eNOS) phosphorylation at Ser1177 in control and Ang II-treated cells. Bars represent means ± s.e.m. of four to six independent experiments. *Po0.05; **Po0.01 vs. the control. ##Po0.01 vs. vehicle. DAF-FM, 4-amino-5-methylamino-2′,7′- difluorofluorescein; DAPI, 4,6-diamidino-2-phenylindole. A full color version of this figure is available at the Hypertension Research journal online.

Article Snippet: After blocking with 5% skim milk, membranes were incubated with purified mouse anti-eNOS (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and purified mouse anti-phospho Ser1177 eNOS (1:1000, Santa Cruz) at 4 °C overnight.

Techniques: Preserving, Western Blot, Phospho-proteomics, Control

Journal: Journal of Applied Physiology

Article Title: Voluntary wheel running prevents salt-induced endothelial dysfunction: role of oxidative stress

doi: 10.1152/japplphysiol.00421.2018

Figure Lengend Snippet: A–C: total endothelial nitric oxide synthase (eNOS) protein expression in the femoral artery normalized to β-actin (A), phosphorylated eNOS (eNOS-P, ser1177) relative to total eNOS (B), and representative Western blots (C). D–F: total eNOS protein expression in the aorta normalized to β-actin (D), eNOS-P (ser1177) relative to total eNOS (E), and representative Western blots (F). Values are means ± SE; n = 9 or 10 per group. Data analysis was performed with a 1-way ANOVA with a Tukey’s post hoc test. HS, high salt; NS, normal salt; Sed, sedentary; VWR, voluntary wheel running. *vs. HS, †vs. NS-VWR (P < 0.05).

Article Snippet: Samples were loaded onto 10% Tris·HCl gels (Thermo Scientific), transferred to nitrocellulose membranes, and stained with primary antibodies (all purchased from Santa Cruz Biotechnology) for the specific protein of interest: endothelial NOS (eNOS, 1:1,000; sc-376751), phosphorylated eNOS (ser1177, 1:1,000; sc-293032), nitrotyrosine (1:1,000; sc-32757), SOD-1 (1:1,000; sc-271014), SOD-2 (1:1,000; sc-137254), catalase (1:500; sc-271803), glutathione peroxidase-1/2 (GPx-1/2, 1:500; sc-133152), NADPH oxidase 4 (Nox4, 1:500; sc-55142), and NADPH oxidase 2/Gp91-phox (1:500; sc-74514).

Techniques: Expressing, Western Blot