Journal: The Journal of Experimental Medicine

Article Title: Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice

doi: 10.1084/jem.20062340

Figure Lengend Snippet: eNOS localization and activation in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries. (A and B) eNOS (A and B, green), Cav-1 (A, red), and GM130 (B, red) expression in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries in situ by whole-mount immunostaining. Bar, 100 nm. Nuclei (blue) are detected via DAPI labeling (C) caveolins, eNOS, HSP90; Akt protein expression and eNOS (Ser 1179 ) and Akt (Ser 473 ) phosphorylation in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries. These blots are representative of four individual experiments. (D) Summary of eNOS and Akt phosphorylation-isolated vessels. Results are shown as phosphoprotein/total protein for eNOS and Akt, respectively. *, P, < 0.05, compared with WT vessels; †, P < 0.05, compared with Cav-1 KO vessels. Data are expressed as the mean ± the SEM.

Article Snippet: The membranes were probed with rabbit anti–Cav-1 antibody (BD Transduction Laboratories), mouse anti–Cav-2 antibody (BD Transduction Laboratories), mouse anti–Cav-3 antibody (BD Transduction Laboratories), mouse anti–β-actin antibody (Sigma-Aldrich), mouse anti-eNOS antibody (BD Transduction Laboratories), rabbit anti–phospho-eNOS (Ser 1179 ) antibody (BD Transduction Laboratories), mouse anti-HSP90 antibody (BD Transduction Laboratories), rabbit anti-Akt antibody (Cell Signaling Technology), rabbit anti–phospho-Akt (Ser 473 ) antibody (Cell Signaling Technology), rabbit anti-p42/44 antibody (Cell Signaling Technology), rabbit anti–phospho-p42/44 (Thr 202 /Tyr 204 ) antibody (Cell Signaling Technology), rabbit anti-p38 antibody (Cell Signaling Technology), rabbit anti–phospho-p38 (Thr 180 /Tyr 182 ) antibody (Cell Signaling Technology), rabbit anti-JNK antibody (Cell Signaling Technology), and rabbit anti–phospho-JNK (Thr 183 /Tyr 185 ) antibody (Cell Signaling Technology) at dilutions ranging from 1:200 to 1:1,000.

Techniques: Activation Assay, Expressing, In Situ, Immunostaining, Labeling, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice

doi: 10.1084/jem.20062340

Figure Lengend Snippet: Enhanced Akt and p42/44 ERK phosphorylation and fibrogenic gene expression in hearts and lungs of WT, Cav-1 KO, and Cav-1 RC mice. (A and B) Total caveolins, eNOS, HSP90, Akt, p42/44, p38, and JNK protein expression and phosphor-Akt (Ser 473 ), p42/44 (Thr 202 /Tyr 204 ), p38 (Thr 180 /Tyr 182 ) in tissue extracts. (A) Typical Western blots from six individual experiments; (B) the summary of these experiments by quantitative imaging. Akt (Ser 473 ) phosphorylation (C) and p42/44 (Thr 202 /Tyr 204 ) phosphorylation (D) in main coronary arteries of the three strains of mice, respectively. Akt (Ser 473 ) phosphorylation (E) and p42/44 (Thr 202 /Tyr 204 ) phosphorylation (F) in pulmonary arteries (top) and lung parenchyma (middle showing large airway and bottom showing alveoli). Photos are representative from four separate experiments. Quantitative analysis of PECAM-1 and E-cad (G) and total nuclei (H) in hearts and lungs of WT, Cav-1 KO, and Cav-1 RC mice. Expression of TGF-β1, and interstitial collagen genes (I) by RT-PCR (compared with GAPDH) in total RNA isolated from hearts and lungs of WT, Cav-1 KO, and Cav-1 RC mice. Data are the means ± the SEM. n = 4 mice per group.

Article Snippet: The membranes were probed with rabbit anti–Cav-1 antibody (BD Transduction Laboratories), mouse anti–Cav-2 antibody (BD Transduction Laboratories), mouse anti–Cav-3 antibody (BD Transduction Laboratories), mouse anti–β-actin antibody (Sigma-Aldrich), mouse anti-eNOS antibody (BD Transduction Laboratories), rabbit anti–phospho-eNOS (Ser 1179 ) antibody (BD Transduction Laboratories), mouse anti-HSP90 antibody (BD Transduction Laboratories), rabbit anti-Akt antibody (Cell Signaling Technology), rabbit anti–phospho-Akt (Ser 473 ) antibody (Cell Signaling Technology), rabbit anti-p42/44 antibody (Cell Signaling Technology), rabbit anti–phospho-p42/44 (Thr 202 /Tyr 204 ) antibody (Cell Signaling Technology), rabbit anti-p38 antibody (Cell Signaling Technology), rabbit anti–phospho-p38 (Thr 180 /Tyr 182 ) antibody (Cell Signaling Technology), rabbit anti-JNK antibody (Cell Signaling Technology), and rabbit anti–phospho-JNK (Thr 183 /Tyr 185 ) antibody (Cell Signaling Technology) at dilutions ranging from 1:200 to 1:1,000.

Techniques: Expressing, Western Blot, Imaging, Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 1. Characterization of rabbit EPCs and comparison with HUVECs. A, B, Cobblestone morphology of EPCs and HUVEC 10 days after plating (40); C, D, immunohistochemical detec- tion of CD31; immunofluorescence detection of (E, F), VE-cadherin; (G, H), eNOS; (I, J), vWF. Preparations were viewed at 400 unless otherwise indicated. Preparations in G, H, I, and J were counterstained with Hoechst 33342.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Comparison, Immunohistochemical staining

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 2. Ex vivo retroviral transduction and transgene expression in EPCs. A, Confluent monolayer of EPCs trans- duced with MSCV retrovirus expressing GFP viewed under normal (white) light (100); B, view of same field under green fluorescent light; C, D, human eNOS transgene mRNA and protein expression in untransduced and trans- duced EPCs, respectively; E, F, HO-1 mRNA and protein expression in untransduced and transduced EPCs, respectively.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Ex Vivo, Retroviral, Transduction, Expressing

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 3. In vivo transgene expression after transplantation of EPCs onto injured carotid arteries. A, Immunohistochemical detection of eNOS (arrows) in frozen sections from denuded vessel trans- planted with eNOS-EPCs (250). B, eNOS immunoreactivity in sections from control vessel transplanted with GFP- EPCs (250); C, immunohistochemical detection of human HO-1 (arrows) in fro- zen sections from vessel transplanted with HO-1-EPCs (250); D, control ves- sels transplanted with GFP-EPCs (250); E, detection of human eNOS transgene mRNA expression by RT-PCR; F, human eNOS protein expression in homoge- nates from eNOS-EPC–transplanted ves- sels; G, human HO-1 transgene mRNA expression; H, HO-1 protein expression in homogenates from HO-1-EPC–trans- planted vessels. GFP-EPC–transplanted vessels served as controls.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: In Vivo, Expressing, Transplantation Assay, Immunohistochemical staining, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 4. Reendothelialization of injured arteries after transplan- tation of EPCs expressing GFP, eNOS, or HO-1 genes. Low- power (100) magnification of vessel profiles stained with anti- CD31 antibody in A, unseeded; B, GFP-EPCs; C, eNOS-EPCs; and D, HO-1-EPCs. Red CD31-positive staining indicates pres- ence of an endothelial monolayer. E, Quantification of endotheli- alization by planar morphometry. Endothelialization was enhanced in EPC-transplanted vessels compared with unseeded saline-treated controls (*P0.05, n4/group).

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Expressing, Staining, Saline

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 5. Inhibition of neointimal proliferation by EPCs in injured carotid arteries. All sections were stained with Accustain elastic stain. Low (40, left) and high (200, right) magnification of vessel profiles. A, B, Saline-injured artery transplanted with eNOS-EPCs (n6); C, D, balloon-injured artery transplanted with EPC expressing GFP (n6); E, F, balloon-injured artery transplanted with EPC expressing eNOS (n6); G, H, balloon- injured artery transplanted with HO-1-EPCs (n6); I, neointima/ media ratios in unseeded and EPC-transplanted injured vessels. Arrows indicate neointima. *Saline vs GFP-EPCs, eNOS-EPCs, HO-1-EPCs; †eNOS-EPCs vs GFP-EPCs, P0.05.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Inhibition, Staining, Saline, Expressing

Journal: Biomolecules

Article Title: Lymphocyte-Specific Protein 1 Regulates Expression and Stability of Endothelial Nitric Oxide Synthase

doi: 10.3390/biom14010111

Figure Lengend Snippet: eNOS and LSP1 interaction in human EA.hy926 endothelial cells. ( A ) Representative immunoblotting of eNOS immunoprecipitate. EA.hy926 cells were lysed in CHAPS IP lysis buffer and clarified protein extract was incubated with anti-eNOS rabbit polyclonal antibody or isotype control IgG. Immunoprecipitated proteins were subjected to western blotting analysis with mouse anti-eNOS monoclonal antibody (upper blot) and rabbit anti-LSP1 polyclonal antibody (lower blot). Data are representative of at least four independent experiments. ( B ) Reverse co-immunoprecipitation reveals the association of LSP1 with wild-type (WT) 140 kDa eNOS and truncated 100 kDa eNOS. Data are representative of at least two independent experiments. ( C ) Co-immunoprecipitation of LSP1-tGFP and eNOS. EA.hy926 cells stably transfected with tGFP-LSP1 construct were lysed in Pierce IP lysis buffer and purified protein extracts were subjected to immunoprecipitation by anti-tGFP mouse monoclonal antibody or non-immune mouse IgG. Fractions (input, IP elute, and post-IP supernatant) were resolved by SDS-PAGE electrophoresis and labelled with anti-eNOS mouse monoclonal antibody (top), and mouse anti-turbo GFP monoclonal antibody (middle), and anti-GAPDH mouse monoclonal antibody (bottom). Blots representative of three experiments are shown.

Article Snippet: The clarified protein extracts were analyzed through immunoblotting for the detection of eNOS and immunoblots were probed with mouse monoclonal anti-eNOS antibodies against the C-terminus of the enzyme (BD Bioscience), mouse monoclonal anti-β-actin antibody (Santa Cruz), and rabbit polyclonal anti-eNOS antibody (Abnova, Taipei, Taiwan; # PAB12680).

Techniques: Western Blot, Lysis, Incubation, Control, Immunoprecipitation, Stable Transfection, Transfection, Construct, Purification, SDS Page, Electrophoresis

Journal: Biomolecules

Article Title: Lymphocyte-Specific Protein 1 Regulates Expression and Stability of Endothelial Nitric Oxide Synthase

doi: 10.3390/biom14010111

Figure Lengend Snippet: ( A ) Colocalization of native LSP1 and eNOS in human macrovascular ECs. EA.hy926 cells were cultured on poly D-lysine treated tissue culture glass slides and were fixed, permeabilized, and immunolabelled with mouse monoclonal anti-eNOS (green) and rabbit polyclonal anti-LSP1 (red) primary antibodies, followed by detection with Alex-Fluor-488-labelled goat anti-mouse and Alexa-Fluor-647-labelled donkey anti-rabbit secondary fluorescent probes. Note the typical localization of eNOS to perinuclear region and distinct plasma membrane pools, with fainter staining in cytosol. Arrow denotes significant colocalization (yellow) of eNOS and LSP1 in the composite image, particularly in perinuclear region. Immunofluorescence images were captured by confocal microscope using 63× oil lens. ( B ) Colocalization of exogenous LSP1-tGFP and eNOS in human macrovascular ECs. EA.hy926 cells stably transfected with LSP1-tGFP construct were exclusively immunolabelled with mouse monoclonal antibody to eNOS (red), followed by identification with Alexa-Fluor-568-conjugated donkey anti-mouse secondary antibody. Arrows indicate areas of colocalization of the two proteins in the composite image (yellow). Turbo-GFP tagged human LSP1 colocalizes with eNOS in distinct perinuclear distribution. The pattern of colocalization of exogenous LSP1 is consistent with native LSP1 colocalization with eNOS. The blue fluorescence represents EC nucleus staining with DAPI. The images were acquired by Zeiss LSM700 inverted confocal microscope using 40× oil lens.

Article Snippet: The clarified protein extracts were analyzed through immunoblotting for the detection of eNOS and immunoblots were probed with mouse monoclonal anti-eNOS antibodies against the C-terminus of the enzyme (BD Bioscience), mouse monoclonal anti-β-actin antibody (Santa Cruz), and rabbit polyclonal anti-eNOS antibody (Abnova, Taipei, Taiwan; # PAB12680).

Techniques: Cell Culture, Membrane, Staining, Immunofluorescence, Microscopy, Stable Transfection, Transfection, Construct, Fluorescence

Journal: Biomolecules

Article Title: Lymphocyte-Specific Protein 1 Regulates Expression and Stability of Endothelial Nitric Oxide Synthase

doi: 10.3390/biom14010111

Figure Lengend Snippet: eNOS expression in vascular tissues from WT and LSP KO mice. ( A ) eNOS dimerization and dissociation of eNOS dimers to 100 kDa fragments in vivo in LSP1-KO aortae. eNOS was immunoprecipitated from the WT and LSP1-KO aorta tissue lysates followed by immunoblotting with mouse anti-eNOS monoclonal Ab. The experiment was repeated two times with similar results, n = 2. IgG HC is immunoglobulin heavy chain. ( B ) eNOS instability in vivo in LSP1-KO aortae. Original uncropped immunoblot of crude aortic tissue lysates subjected to LT non-denaturing SDS-PAGE, probed with mouse anti-eNOS monoclonal Ab. Prominent 100 kDa, and faint 75 and 70 kDa eNOS immunoreactive bands were only observed in LSP1-KO aorta lane. ( C ) eNOS protein expression in aortae from age-matched WT and LSP1 KO mice. Immunoblots shown are representative of four independent experiments, n = 4. ( D ) eNOS cleavage in vascular tissues of LSP1 KO mice. Aortae and mesenteric arteries from WT and LSP1-KO mice were lysed in CHAPS lysis buffer. The immunoblots were probed with rabbit anti-eNOS polyclonal antibody (top), and GAPDH (bottom). The rabbit anti-eNOS polyclonal antibody has revealed 75 kDa and 70 kDa immunoreactive bands which were the cleavage fragments of eNOS. The 75 and 70 kDa bands were recognized as eNOS by LC-MS/MS analysis. Data are representative of three independent experiments. Lane 1 and Lane 2 represent aorta protein samples, whereas Lane 3 and Lane 4 represent mesenteric arterial lysates. Arrowheads in all immunoblots indicate eNOS truncated fragments. Graphs demonstrate the quantification of eNOS dimer-to-eNOS 100 kDa ratio ( E ), and the relative eNOS protein abundance in aortae ( F ) and mesenteric arteries ( G ) of the WT and LSP1-KO mice. The results are displayed as mean ± SEM. * denotes p value < 0.05; ** denotes p < 0.01 according to one-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: The clarified protein extracts were analyzed through immunoblotting for the detection of eNOS and immunoblots were probed with mouse monoclonal anti-eNOS antibodies against the C-terminus of the enzyme (BD Bioscience), mouse monoclonal anti-β-actin antibody (Santa Cruz), and rabbit polyclonal anti-eNOS antibody (Abnova, Taipei, Taiwan; # PAB12680).

Techniques: Expressing, In Vivo, Immunoprecipitation, Western Blot, SDS Page, Lysis, Liquid Chromatography with Mass Spectroscopy

Journal: Molecular neurobiology

Article Title: Autologous bone marrow mononuclear cell transplantation delays progression of carotid atherosclerosis in rabbits

doi: 10.1007/s12035-015-9347-3

Figure Lengend Snippet: BMMNC transplantation reverses atherosclerosis-induced changes in antioxidant enzymes, eNOS, inflammatory response, and cell apoptosis. (A) Western blot analysis of eNOS in plaques. (B) Quantification analysis showed that plaques from the BMMNC group contained more eNOS than did plaques from the vehicle group. (C–F) BMMNC transplantation upregulated expression of the antioxidant enzymes GPx-1 and SOD-1 in plaque. (C, E) Western blot analysis of GPx-1 and SOD-1 in plaque. (D, F) Quantification of band densities showed that plaques from rabbits treated with BMMNCs had significantly higher GPx-1 and SOD-1 protein expression than did plaques from vehicle-treated rabbits. (G–H) BMMNC transplantation reduced expression of cell-death marker cleaved caspase-3 in plaque. (G) Western blot analysis of cleaved caspase-3. (H) Quantification analysis showed that cleaved caspase-3 was significantly lower in plaques from BMMNC-treated rabbits than in plaques from vehicle-treated rabbits. *p<0.05 vs. vehicle group. (I–L) BMMNC transplantation significantly decreased the mRNA expression of proinflammatory cytokines in plaque. (I) Representative RT-PCR bands showing mRNA expression of IL-1 β, TNF-α, and MMP-9 in plaque. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (J–L) Analysis of band optical densities showed that BMMNC transplantation significantly decreased the mRNA expression of IL-1 β, TNF-α, and MMP-9 compared with that in vehicle-treated animals. *p<0.05 vs. vehicle group. n=6 rabbits per group.

Article Snippet: The primary antibodies were mouse anti-rabbit IGF-1 (1:200, Global Biological Technologies Inc., Shanghai, China), rabbit anti-rabbit IGF-1R (1:100, Biosynthesis), rabbit anti-rabbit eNOS (1:400, Boster), mouse anti-rabbit GPx-1 (1:100, Abcam), goat anti-rabbit SOD-1 (1:1000, Abcam), rabbit anti-rabbit caspase-3 (1:1000, Abcam), and mouse anti-rabbit β-actin (1:500, Boster).

Techniques: Transplantation Assay, Western Blot, Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Molecular Vision

Article Title: Inhibition by brimonidine of forskolin-induced nitric oxide synthase expression in human ciliary bodies in vitro

doi:

Figure Lengend Snippet: Primers used in RT-PCR analysis of NOS isoforms.

Article Snippet: The membrane was incubated with anti-human nNOS (1:2000 dilution), iNOS (1:200 dilution), or eNOS (1:1000 dilution) rabbit polyclonal antibodies (Boster Biotechnology Co. Ltd, Wuhan, China).

Techniques: Sequencing

Journal: Molecular Vision

Article Title: Inhibition by brimonidine of forskolin-induced nitric oxide synthase expression in human ciliary bodies in vitro

doi:

Figure Lengend Snippet: mRNA expression of three NOS isoforms (nNOS, iNOS, and eNOS) in human ciliary bodies. RT-PCR analysis of NOS mRNAs are shown in A . Each experiment was repeated 3 times and the mean values of the results of these experiments are shown in B . The asterisk indicates a p<0.001, versus forskolin (0 h) and the double asterisk indicates a p<0.05, versus forskolin (24 h).

Article Snippet: The membrane was incubated with anti-human nNOS (1:2000 dilution), iNOS (1:200 dilution), or eNOS (1:1000 dilution) rabbit polyclonal antibodies (Boster Biotechnology Co. Ltd, Wuhan, China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Vision

Article Title: Inhibition by brimonidine of forskolin-induced nitric oxide synthase expression in human ciliary bodies in vitro

doi:

Figure Lengend Snippet: Protein expression of three NOS isoforms (nNOS, iNOS, and eNOS) in human ciliary bodies. Western blot analysis of NOS proteins are shown in A . Each experiment was repeated 3 times and the mean values of the results of these experiments are shown in B . The asterisk denotes a p<0.01, versus forskolin (0 h) and the double asterisk indicates a p<0.05, versus forskolin (24 h).

Article Snippet: The membrane was incubated with anti-human nNOS (1:2000 dilution), iNOS (1:200 dilution), or eNOS (1:1000 dilution) rabbit polyclonal antibodies (Boster Biotechnology Co. Ltd, Wuhan, China).

Techniques: Expressing, Western Blot

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: PLoS ONE

Article Title: Jujuboside B Reduces Vascular Tension by Increasing Ca 2+ Influx and Activating Endothelial Nitric Oxide Synthase

doi: 10.1371/journal.pone.0149386

Figure Lengend Snippet: (A)Jujuboside B increased NO generation in HAECs concentration-dependently. * P < 0.05, ** P < 0.01 compared with Control, n = 3. (B) Jujuboside B increased NO generation in HAECs negative time-dependently. * P < 0.05, ** P < 0.01 compared with 15 min group, n = 3. (C) L-NAME significantly inhibited Jujuboside B-induced NO generation. * P < 0.05, ** P < 0.01, n = 3. (D) L-NAME significantly inhibited Jujuboside B-induced eNOS activation. ** P < 0.01, ## P < 0.01, n = 4. (E) Western blot analysis of eNOS and p-eNOS at Serine-1177 in HAECs showed that Jujuboside B significantly increased p-eNOS Serine-1177 expression. (F) Densitometric analysis showed that Jujuboside B significantly increased p-eNOS Serine-1177 expression. * P < 0.05 compared with Control, n = 3.

Article Snippet: The residual protein bands were blocked with 5% milk and the membrane was incubated with rabbit p-eNOS (Serine-1177) antibody and rabbit eNOS antibody (Cell Signaling Technology, Inc, Boston, USA) dilution overnight.

Techniques: Concentration Assay, Control, Activation Assay, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Jujuboside B Reduces Vascular Tension by Increasing Ca 2+ Influx and Activating Endothelial Nitric Oxide Synthase

doi: 10.1371/journal.pone.0149386

Figure Lengend Snippet: (A)EGTA and SKF96365 significantly inhibited Jujuboside B-induced eNOS activation. ** P < 0.01 compared with Control, # P < 0.05, ## P <0.01 compared with Jujuboside B, n = 4. (B) Western blot analysis showed that EGTA and SKF96365 significantly inhibited Jujuboside B-induced eNOS phosphorylation at Serine-1177. (C) Densitometric analysis showed that EGTA and SKF96365 significantly inhibited Jujuboside B-induced eNOS phosphorylation at Serine-1177. # P < 0.05 compared with Control, * P < 0.05 compared with Jujuboside B, n = 3.

Article Snippet: The residual protein bands were blocked with 5% milk and the membrane was incubated with rabbit p-eNOS (Serine-1177) antibody and rabbit eNOS antibody (Cell Signaling Technology, Inc, Boston, USA) dilution overnight.

Techniques: Activation Assay, Control, Western Blot, Phospho-proteomics

Journal: American Journal of Translational Research

Article Title: PKCβ increases ROS levels leading to vascular endothelial injury in diabetic foot ulcers

doi:

Figure Lengend Snippet: Expression changes in PKCβ/p66shc and inflammation-related proteins in diabetic foot ulcer. A, B. Western blot analysis showed significantly increased expression levels of p-PKCβ and p-P66shc in the DFU group vs. the CON group (P<0.01). C, D. Western blot analysis showed decreased protein levels of eNOS and significantly increased expression levels of ICAM-1 and p-NF-κB in the DFU group (P<0.01). n≥3 per group, with the experiment repeated three times. Values are the means and standard errors (*P<0.05 and **P<0.01 versus CON).

Article Snippet: Antibodies against endothelial nitric oxide synthase (eNOS) (35362), ICAM-1 (4915), nuclear factor-kB (NF-κB) (49D7), p-NF-κB (108D2), and β-actin (13E5) were bought from Cell Signaling Technology (Shanghai, China).

Techniques: Expressing, Western Blot

Journal: American Journal of Translational Research

Article Title: PKCβ increases ROS levels leading to vascular endothelial injury in diabetic foot ulcers

doi:

Figure Lengend Snippet: LY333531 inhibited changes in the activity of the PKCβ-p66shc pathway caused by high glucose in HUVECs. A, B. LY333531 rescued the increased levels of p-PKCβ and p-P66shc in HUVECs treated with glucose. C, D. LY333531 rescued the increased levels of ICAM-1 and p-NF-κB and the inhibition of eNOS in HUVECs treated with glucose. E, F. Immunofluorescence showed that LY333531 could restore the expression levels and distribution of p-P66shc and restore the eNOS level in HUVECs treated with high glucose. G, H. The mean fluorescence intensity of p-P66shc and eNOS was quantified and is presented as the means ± SDs. Each group was a mixture of cells collected three times (n = 3), and the experiment was repeated three times. Values are the means and standard errors (*P<0.05 and **P<0.01 versus CON).

Article Snippet: Antibodies against endothelial nitric oxide synthase (eNOS) (35362), ICAM-1 (4915), nuclear factor-kB (NF-κB) (49D7), p-NF-κB (108D2), and β-actin (13E5) were bought from Cell Signaling Technology (Shanghai, China).

Techniques: Activity Assay, Inhibition, Immunofluorescence, Expressing, Fluorescence