Journal: Circulation Research

Article Title: Apurinic/Apyrmidinic Endonuclease 1 Regulates Endothelial NO Production and Vascular Tone

doi: 10.1161/01.res.0000146947.84294.4c

Figure Lengend Snippet: Figure 4. APE1/ref-1 stimulates eNOS catalytic activity through a PI3-K/Akt kinase–dependent mechanism by upregulating H-ras expression. a, In vitro NOS catalytic activity at a Ca2 of 0.6 mmol/L measured in lysates of BAECs infected with Adgal, AdAPE1/ ref-1, and AdAktAA. Numbers indicate moi of each virus. *P0.05 compared with all other conditions. b, Representa- tive Western blots for total Akt, P-S473- Akt, P-S1177-eNOS, APE1/ref-1, and HA-p85 in lysates of HUVECs infected with Adgal, AdAPE1/ref-1, and Adp85 (adenovirus encoding an HA-tagged dominant-inhibitory form of the p85 regu- latory subunit of PI3-K). Phospho-GSK-3 blot indicates in vitro phosphorylation of glycogen synthase kinase-3 by immuno- precipitated active Akt from whole-cell lysates. All viruses were used at 200 moi. c, In vitro NOS catalytic activity at titrated concentrations of free Ca2 (pCa Log-free Ca2) in lysates of COS-7 cells cotransfected with eNOS and (1) APE1/ref-1, or (2) APE1/ref-1H-rasN17. *P0.05 compared with all other condi- tions d, In vitro NOS catalytic activity in lysates of COS-7 cells cotransfected with eNOS and (1) APE1/ref-1, or (2) APE1/ref-1 (C65/93A). Free Ca2 concentration in reaction buffer was titrated to 106 mol/L (n3). *P0.05 compared with all other conditions. e, left, Total and GTP-bound H-ras in lysates of COS-7 cells trans- fected with control plasmid, APE1/ref-1, or APE1/ref-1(C65/93A). Densitometric quantification of total and GTP–H-ras is shown at bottom. Right, H-ras, rac1, and APE1/ref-1 expression in HUVECs infected with Adgal or AdAPE1/ref-1 at 200 moi each. Comparison of densito- metric values is shown at bottom. *P0.05 compared with Adgal. f, Expression of total H-ras, total Akt, P-S473-Akt, total eNOS, and P-S1177- eNOS in extracts of homogenized whole aortas from APE1/ref-1/ and WT mice. Bottom, Comparison of densitometric values of H-ras and P-S1177–eNOS is shown at bottom. *P0.05 compared with WT.

Article Snippet: Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000146947.84294.4c 902 by guest on May 17, 2015http://circres.ahajournals.org/Downloaded from Twenty-four to 48 hours after transfection with DNA or infection with adenoviruses, expression and phosphorylation of proteins (50 g) were determined by immunoblotting with antibodies to eNOS (SC-654; Santa Cruz Biotechnology), phosphoserine-1177 eNOS (phosphoserine-1179 for bovine eNOS), (9571; Cell Signaling), inducible NOS (iNOS; SC-650; Santa Cruz Biotechnology), APE1/ ref-1 (SC-5572; Santa Cruz Biotechnology), Akt (9272; Cell Signaling), H-ras (SC-520; Santa Cruz Biotechnology), rac1 (05 to 389; Upstate Biotechnology), phosphoserine-473 Akt (9271; Cell Signaling), and hemagglutinin (HA) (1583816; Roche).

Techniques: Activity Assay, Expressing, In Vitro, Infection, Virus, Western Blot, Phospho-proteomics, Concentration Assay, Control, Plasmid Preparation, Comparison

Journal: Marine Drugs

Article Title: The Role of Sargahydroquinoic Acid and Sargachromenol in the Anti-Inflammatory Effect of Sargassum yezoense

doi: 10.3390/md22030107

Figure Lengend Snippet: Effect of SYEE on LPS-stimulated RAW 264.7 macrophages. ( a ) Effect of SYEE on cell viability of RAW 264.7 macrophages were measured. ( b ) Levels of mRNA expression of Tnf , Il6 , Cox2 , Nos2 , and Nox2 were presented as fold changes relative to the LPS-stimulated samples; ( c ) Level of protein expression of COX2 and NOS2 were presented as fold changes relative to the LPS-stimulated samples. β-Actin (ACTB) was used as the internal control. ( d ) Level of secreted TNF was presented as fold changes relative to the LPS-stimulated samples. The data are expressed as mean ± standard deviation ( n = 3). Columns without a common letter differ from each other significantly ( p < 0.05).

Article Snippet: The antibodies against NOS2 and COX2 (sc-650 and sc-1745, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used, and beta-actin (sc-47778, 1:1000, Santa Cruz Biotechnology) served as a loading control for data normalization.

Techniques: Expressing, Control, Standard Deviation

Journal: Marine Drugs

Article Title: The Role of Sargahydroquinoic Acid and Sargachromenol in the Anti-Inflammatory Effect of Sargassum yezoense

doi: 10.3390/md22030107

Figure Lengend Snippet: Effect of SYEE on LPS- and IFN-γ-stimulated mouse BMDMs. Level of mRNA expression of Il1b , Cox2 , Nos2 , Nox1 , Nox2 , and Cd86 were presented as fold changes relative to the LPS-stimulated samples. The data are expressed as mean ± standard deviation ( n = 3). Columns without a common letter differ from each other significantly ( p < 0.05).

Article Snippet: The antibodies against NOS2 and COX2 (sc-650 and sc-1745, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used, and beta-actin (sc-47778, 1:1000, Santa Cruz Biotechnology) served as a loading control for data normalization.

Techniques: Expressing, Standard Deviation

Journal: Marine Drugs

Article Title: The Role of Sargahydroquinoic Acid and Sargachromenol in the Anti-Inflammatory Effect of Sargassum yezoense

doi: 10.3390/md22030107

Figure Lengend Snippet: Effect of SYHF, SYCF, and SYEtF on LPS-stimulated RAW 264.7 macrophages. The effect of ( a ) SYHF, ( b ) SYCF, and ( c ) SYEtF on cell viablity of RAW 264.7 macrophages. The effect of ( d ) SYHF, ( e ) SYCF, and ( f ) SYEtF on mRNA expression of Tnf , Il6 , Cox2 , Nos2 , and Nox2 . The data are expressed as mean ± standard deviation ( n = 3). Columns without a common letter differ from each other significantly ( p < 0.05).

Article Snippet: The antibodies against NOS2 and COX2 (sc-650 and sc-1745, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used, and beta-actin (sc-47778, 1:1000, Santa Cruz Biotechnology) served as a loading control for data normalization.

Techniques: Expressing, Standard Deviation

Journal: Marine Drugs

Article Title: The Role of Sargahydroquinoic Acid and Sargachromenol in the Anti-Inflammatory Effect of Sargassum yezoense

doi: 10.3390/md22030107

Figure Lengend Snippet: Contribution of SHQA and SCM on inhibition of LPS-induced inflammation. ( a ) The effect of SYCF, and corresponding concentration of SHQA and SCM on mRNA expression of Tnf , Il1b , Il6 , Nos2 , Cox2 , and Nox2 . ( b ) The effect of SYCF, and corresponding concentration of SHQA and SCM on protein expression of NOS2 and COX2. The data are expressed as mean ± standard deviation ( n = 3). Columns without a common letter differ from each other significantly ( p < 0.05).

Article Snippet: The antibodies against NOS2 and COX2 (sc-650 and sc-1745, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used, and beta-actin (sc-47778, 1:1000, Santa Cruz Biotechnology) served as a loading control for data normalization.

Techniques: Inhibition, Concentration Assay, Expressing, Standard Deviation

Journal: Journal of Inflammation (London, England)

Article Title: Antioxidant activity of tuberosin isolated from Pueraria tuberose Linn

doi: 10.1186/1476-9255-7-47

Figure Lengend Snippet: Effect of tuberosin on LPS induced NO production and iNOS expression by attached rat peritoneal macrophages.

Article Snippet: Finally, the blot was incubated with rabbit polyclonal anti-iNOS antibody (SC650, Santa Cruz Biotechnology, 1/1000 in TBS-Tween-20 buffer) at 4°C overnight and visualized by alkaline phosphatase-conjugated anti-rabbit IgG as the secondary antibody.

Techniques: Expressing, Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: Antioxidant activity of tuberosin isolated from Pueraria tuberose Linn

doi: 10.1186/1476-9255-7-47

Figure Lengend Snippet: Effect of different concentrations of Tuberosin on LPS induced iNOS expression in attached rat peritoneal macrophages . The macrophages were pretreated with quercetin and tuberosin as given below for 30 minutes and then LPS was added (20 ng/ml) and incubated for 17 hrs. The normal cells were exposed to 0.1% DMSO without any LPS. Lane-1: LPS(20 ng/ml),Lane-2: Normal cells. Lane3:LPS+Quercetine(50 ng/ml), Lane4:LPS+Tuberosine(100 ng/ml), Lane-5: LPS+Tuberosine(300 ng/ml), Lane-6: LPS+Tuberosine(600 ng/ml). The bars depict densitometric analysis of western blot (given in the inset). This picture represents one out of total three experiments carried out separately.

Article Snippet: Finally, the blot was incubated with rabbit polyclonal anti-iNOS antibody (SC650, Santa Cruz Biotechnology, 1/1000 in TBS-Tween-20 buffer) at 4°C overnight and visualized by alkaline phosphatase-conjugated anti-rabbit IgG as the secondary antibody.

Techniques: Expressing, Incubation, Western Blot

Journal:

Article Title: Gamma Interferon Prevents the Inhibitory Effects of Oxidative Stress on Host Responses to Escherichia coli Infection

doi: 10.1128/IAI.69.4.2621-2629.2001

Figure Lengend Snippet: Glutathione depletion differentially inhibits hepatic inflammatory responses in mice challenged with 5 × 107 E. coli bacteria. All injections were by the i.p. route. Immunohistology was used to detect the expression of specific proteins in the livers of the following groups of mice. (A) HSP-32 staining in mice 5 h after the injection of DEM (see Fig. ​Fig.1A).1A). Note the expression of HSP-32 by Kupffer cells (solid arrow), hepatocytes (open arrow) and hepatic sinusoidal endothelial cells (arrowhead). (B) TNF-α staining in Kupffer cells (solid arrow) 1.5 h after vehicle-treated mice were challenged with E. coli bacteria. (C) TNF-α staining 1.5 h after DEM-treated mice were challenged with E. coli. Note the absence of TNF-α expression. (D) iNOS staining of Kupffer cells (solid arrow), hepatocytes (open arrow), and hepatic sinusoidal endothelial cells (arrowhead) 6 h after challenging vehicle-treated mice with E. coli. (E) iNOS staining in DEM-treated mice challenged with E. coli. (F) ICAM-1 staining of Kupffer cells (solid arrow) and endothelial cells (arrowhead) 6 h after challenge of the vehicle control group with E. coli. (G) ICAM-1 staining in DEM-treated, E. coli-challenged mice. (H) iNOS staining in DEM-treated mice that were challenged with 4 mg of LPS/kg of body weight showing positive Kupffer cells (solid arrow) and sinusoidal endothelial cells (arrowhead). (I) iNOS staining in DEM-treated mice that were challenged with 4 mg LPS plus 80 μg of recombinant IFN-γ per kg of body weight. Note the staining of all three hepatic cell types, which is similar to that seen in infected mice (panel E above). (J) ICAM-1 staining in DEM-treated mice that were challenged with LPS. (K) ICAM-1 staining in DEM-treated mice challenged with LPS and recombinant IFN-γ. The intense staining is similar that seen in infected mice (panel G above). (L) ICAM-1 staining of Kupffer cells (solid arrow) and endothelial cells (arrowhead) in wild-type BALB/c mice infected with E. coli. (M) ICAM-1 staining in wild-type BALB/c mice treated with DEM and then infected with E. coli. Note the similar staining pattern as in panel L. (N) ICAM-1 staining in IFN-γ-null mice treated with DEM and challenged with E. coli. Note the presence of sinusoidal leukocytes expressing ICAM-1 (circled). (O) F4/80 staining of Kupffer cells (solid arrow) in IFN-γ-null mice treated with DEM and challenged with E. coli. Sinusoidal leukocytes (circled) are negative. Original magnification, ×250.

Article Snippet: The following antibodies were used in the present study: rat anti-mouse TNF-α (MP6-XT22; PharMingen, San Diego, Calif.), rabbit anti-mouse iNOS (SC-650; Santa Cruz Biotechnology, Santa Cruz, Calif.), goat anti-mouse ICAM-1 (SC-1511; Santa Cruz), rat anti-mouse macrophage F4/80 (a gift of Joan Hunt, University of Kansas Medical Center), rat anti-mouse CD14 (rmC5-3; PharMingen), rabbit anti-rat heat shock protein-32 (HSP-32) (SPA-895; StressGen, Victoria, BC, Canada), biotinylated goat anti-rabbit immunoglobulin G (IgG; BioGenex; San Ramon, Calif.), biotinylated rabbit anti-rat IgG (Vector Laboratories; Burlingame, Calif.), and biotinylated donkey anti-goat IgG (Santa Cruz).

Techniques: Expressing, Staining, Injection, Recombinant, Infection

Journal:

Article Title: Gamma Interferon Prevents the Inhibitory Effects of Oxidative Stress on Host Responses to Escherichia coli Infection

doi: 10.1128/IAI.69.4.2621-2629.2001

Figure Lengend Snippet: Comparison of the effects of glutathione depletion on hepatic inflammatory responses to E. coli bacteria and LPS. C3HeB/FeJ mice (six per group) were treated with vehicle or DEM and then challenged i.p. with either 5 × 107 CFU of E. coli bacteria or 100 μg of E. coli LPS. Liver tissues were recovered either 1.5 h (for TNF-α) or 6 h (for iNOS, CD14 or ICAM-1) later. Kupffer cell (KC) and hepatocyte (Hep) responses are expressed as positive cells per HPF (magnification, ×400) (mean ± standard deviation), whereas sinusoidal endothelial cell (EC) responses are expressed as relative staining intensity. Comparable results were obtained with CF1 mice.

Article Snippet: The following antibodies were used in the present study: rat anti-mouse TNF-α (MP6-XT22; PharMingen, San Diego, Calif.), rabbit anti-mouse iNOS (SC-650; Santa Cruz Biotechnology, Santa Cruz, Calif.), goat anti-mouse ICAM-1 (SC-1511; Santa Cruz), rat anti-mouse macrophage F4/80 (a gift of Joan Hunt, University of Kansas Medical Center), rat anti-mouse CD14 (rmC5-3; PharMingen), rabbit anti-rat heat shock protein-32 (HSP-32) (SPA-895; StressGen, Victoria, BC, Canada), biotinylated goat anti-rabbit immunoglobulin G (IgG; BioGenex; San Ramon, Calif.), biotinylated rabbit anti-rat IgG (Vector Laboratories; Burlingame, Calif.), and biotinylated donkey anti-goat IgG (Santa Cruz).

Techniques: Standard Deviation, Staining