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Santa Cruz Biotechnology anti pcna
The effects of cudratricusxanthone A ( 1 ) on IκB-α phosphorylation and degradation ( A ); NF-κB translocation ( B , C ); NF-κB localization as determined by immunofluorescence analysis ( D ); and NF-κB DNA binding activity ( E ) in BV2 microglia. Cells were pretreated for 3 h with the indicated concentrations of cudratricusxanthone A ( 1 ), and stimulated for 1 h with LPS (1 μg/mL). The LPS treatment was performed in the presence of compound. Western blot analyses of IκB-α and p -IκB-α in the cytoplasm ( A ) and NF-κB in the cytoplasm ( B ) and nucleus ( C ) and immunofluorescence analyses ( E ) were carried out as described in the Experimental Section. The band intensity was quantified by densitometry and normalized to <t>β-actin</t> and <t>PCNA,</t> and the values are presented at the bottom of the each band. Relative data represent the means ± SDs of three experiments. * p
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Images

1) Product Images from "A Prenylated Xanthone, Cudratricusxanthone A, Isolated from Cudrania tricuspidata Inhibits Lipopolysaccharide-Induced Neuroinflammation through Inhibition of NF-κB and p38 MAPK Pathways in BV2 Microglia"

Article Title: A Prenylated Xanthone, Cudratricusxanthone A, Isolated from Cudrania tricuspidata Inhibits Lipopolysaccharide-Induced Neuroinflammation through Inhibition of NF-κB and p38 MAPK Pathways in BV2 Microglia

Journal: Molecules

doi: 10.3390/molecules21091240

The effects of cudratricusxanthone A ( 1 ) on IκB-α phosphorylation and degradation ( A ); NF-κB translocation ( B , C ); NF-κB localization as determined by immunofluorescence analysis ( D ); and NF-κB DNA binding activity ( E ) in BV2 microglia. Cells were pretreated for 3 h with the indicated concentrations of cudratricusxanthone A ( 1 ), and stimulated for 1 h with LPS (1 μg/mL). The LPS treatment was performed in the presence of compound. Western blot analyses of IκB-α and p -IκB-α in the cytoplasm ( A ) and NF-κB in the cytoplasm ( B ) and nucleus ( C ) and immunofluorescence analyses ( E ) were carried out as described in the Experimental Section. The band intensity was quantified by densitometry and normalized to β-actin and PCNA, and the values are presented at the bottom of the each band. Relative data represent the means ± SDs of three experiments. * p
Figure Legend Snippet: The effects of cudratricusxanthone A ( 1 ) on IκB-α phosphorylation and degradation ( A ); NF-κB translocation ( B , C ); NF-κB localization as determined by immunofluorescence analysis ( D ); and NF-κB DNA binding activity ( E ) in BV2 microglia. Cells were pretreated for 3 h with the indicated concentrations of cudratricusxanthone A ( 1 ), and stimulated for 1 h with LPS (1 μg/mL). The LPS treatment was performed in the presence of compound. Western blot analyses of IκB-α and p -IκB-α in the cytoplasm ( A ) and NF-κB in the cytoplasm ( B ) and nucleus ( C ) and immunofluorescence analyses ( E ) were carried out as described in the Experimental Section. The band intensity was quantified by densitometry and normalized to β-actin and PCNA, and the values are presented at the bottom of the each band. Relative data represent the means ± SDs of three experiments. * p

Techniques Used: Translocation Assay, Immunofluorescence, Binding Assay, Activity Assay, Western Blot

2) Product Images from "Pioglitazone increases VEGFR3 expression and promotes activation of M2 macrophages via the peroxisome proliferator-activated receptor γ"

Article Title: Pioglitazone increases VEGFR3 expression and promotes activation of M2 macrophages via the peroxisome proliferator-activated receptor γ

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2019.9945

Effect of pioglitazone on the expression of F4/80 and VEGFR3  in vivo . Immunofluorescence analysis of F4/80 and VEGFR3 co-localization (white arrows) in kidney sections from mice in the sham, UUO and UUO + pioglitazone groups. Pio, pioglitazone; UUO, unilateral ureteral obstruction; VEGFR3, vascular endothelial growth factor receptor 3.
Figure Legend Snippet: Effect of pioglitazone on the expression of F4/80 and VEGFR3 in vivo . Immunofluorescence analysis of F4/80 and VEGFR3 co-localization (white arrows) in kidney sections from mice in the sham, UUO and UUO + pioglitazone groups. Pio, pioglitazone; UUO, unilateral ureteral obstruction; VEGFR3, vascular endothelial growth factor receptor 3.

Techniques Used: Expressing, In Vivo, Immunofluorescence, Mouse Assay

3) Product Images from "Anti-Inflammatory Activities of Leaf Oil from Cinnamomum subavenium In Vitro and In Vivo"

Article Title: Anti-Inflammatory Activities of Leaf Oil from Cinnamomum subavenium In Vitro and In Vivo

Journal: BioMed Research International

doi: 10.1155/2019/1823149

Histological observation of rat hind footpads after injecting Carr 0.9% saline (control group) or Carr. (a-d) H E staining of footpad tissue sections from rat in each group. (e-h) iNOS immunohistochemical staining of footpad tissue sections from rat in each group. (i-l) COX-2 immunohistochemical staining of footpad tissue sections from rat in each group. Scale bar = 50 µ m. The infiltrating cells were predominantly neutrophils (N; arrows). The brown staining indicates the interaction of primary and secondary antibodies and the presence of iNOS and COX-2.
Figure Legend Snippet: Histological observation of rat hind footpads after injecting Carr 0.9% saline (control group) or Carr. (a-d) H E staining of footpad tissue sections from rat in each group. (e-h) iNOS immunohistochemical staining of footpad tissue sections from rat in each group. (i-l) COX-2 immunohistochemical staining of footpad tissue sections from rat in each group. Scale bar = 50 µ m. The infiltrating cells were predominantly neutrophils (N; arrows). The brown staining indicates the interaction of primary and secondary antibodies and the presence of iNOS and COX-2.

Techniques Used: Staining, Immunohistochemistry

4) Product Images from "Nimbolide protects against endotoxin-induced acute respiratory distress syndrome by inhibiting TNF-α mediated NF-κB and HDAC-3 nuclear translocation"

Article Title: Nimbolide protects against endotoxin-induced acute respiratory distress syndrome by inhibiting TNF-α mediated NF-κB and HDAC-3 nuclear translocation

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1247-9

Nimbolide interacts with TNF-α and inhibits TNF-α regulated inflammatory signaling. a Docking model of nimbolide in the active site of TNF-α (PDB ID: 2AZ5) and ( b ) its ligand-protein interactions in the binding site of TNF-α. The dark pink dashed lines represent hydrogen bonds. H-bond distances (in Å) between heteroatoms of ligand and amino acid residues are as follows: Ser60 (3.4Å) and Leu120 (3.2Å). The red line indicates arene-arene interaction with Tyr59. A549 cells were pre-treated with nimbolide for 24 h and stimulated by LPS (1 µg/ml) for 12 h. Animals were pre-treated with nimbolide (0.3, 1 and 3 mg/kg) for 5 days later LPS (50 μg) was administered. c TNF-α protein expression was analyzed by confocal microscope. d A 5-µm-sized sections of lung tissues were subjected to IHC to determine TNF-α expression and images were captured at ×400 magnification. Protein expressions of TNF-α, p-p38 MAPK, p-IKK-α/β, p-IκB-α, p-NF-κB, p-GSK-3β, and mTOR were determined by western blotting in ( e ) A549 cells and ( f ) lung tissues, respectively. The HDACs such as HDAC-1, 2, 3, and 4 protein expressions were studied in ( g ) A549 cells and ( h ) lung tissues. i Nimbolide (0.05, 0.1, 1, and 2.5 µM) upon HDAC levels were analyzed by HDAC fluorometric kit and HDAC inhibitory activity was compared with trichostatin A (2.5 µM). Data represented as mean ± SEM (n=3 independent experiments). * P
Figure Legend Snippet: Nimbolide interacts with TNF-α and inhibits TNF-α regulated inflammatory signaling. a Docking model of nimbolide in the active site of TNF-α (PDB ID: 2AZ5) and ( b ) its ligand-protein interactions in the binding site of TNF-α. The dark pink dashed lines represent hydrogen bonds. H-bond distances (in Å) between heteroatoms of ligand and amino acid residues are as follows: Ser60 (3.4Å) and Leu120 (3.2Å). The red line indicates arene-arene interaction with Tyr59. A549 cells were pre-treated with nimbolide for 24 h and stimulated by LPS (1 µg/ml) for 12 h. Animals were pre-treated with nimbolide (0.3, 1 and 3 mg/kg) for 5 days later LPS (50 μg) was administered. c TNF-α protein expression was analyzed by confocal microscope. d A 5-µm-sized sections of lung tissues were subjected to IHC to determine TNF-α expression and images were captured at ×400 magnification. Protein expressions of TNF-α, p-p38 MAPK, p-IKK-α/β, p-IκB-α, p-NF-κB, p-GSK-3β, and mTOR were determined by western blotting in ( e ) A549 cells and ( f ) lung tissues, respectively. The HDACs such as HDAC-1, 2, 3, and 4 protein expressions were studied in ( g ) A549 cells and ( h ) lung tissues. i Nimbolide (0.05, 0.1, 1, and 2.5 µM) upon HDAC levels were analyzed by HDAC fluorometric kit and HDAC inhibitory activity was compared with trichostatin A (2.5 µM). Data represented as mean ± SEM (n=3 independent experiments). * P

Techniques Used: Binding Assay, Expressing, Microscopy, Immunohistochemistry, Western Blot, Activity Assay

Nimbolide inhibits TNF-α regulated NF-κB and HDAC-3 protein expression and nuclear translocation. A549 cells were treated with nimbolide (2.5 µM) for 24 h and stimulated with TNF-α (10 ng/ml) for 30 min. a NF-κB and HDAC-3 protein levels were observed in both cytosolic and nuclear fraction of A549 cells by western blotting. b The confocal analysis was performed to determine the protein expressions of NF-κB and HDAC-3. All the images were captured at ×400 magnification. BEAS-2B cells were transfected with TNF-α and scrambled siRNA and incubated for 24 h. In another set of the experimental group, cells were pre-treated with nimbolide (2.5 µM) for 24 h. Then, cells were stimulated with TNF-α (10 ng/ml) for 30 min except for nimbolide alone (NIM) group (2.5 µM). The expression of TNF-α, NF-κB and HDAC-3 levels were measured by ( c ) western blotting and ( d ) confocal analysis. The images were captured at ×400 magnification
Figure Legend Snippet: Nimbolide inhibits TNF-α regulated NF-κB and HDAC-3 protein expression and nuclear translocation. A549 cells were treated with nimbolide (2.5 µM) for 24 h and stimulated with TNF-α (10 ng/ml) for 30 min. a NF-κB and HDAC-3 protein levels were observed in both cytosolic and nuclear fraction of A549 cells by western blotting. b The confocal analysis was performed to determine the protein expressions of NF-κB and HDAC-3. All the images were captured at ×400 magnification. BEAS-2B cells were transfected with TNF-α and scrambled siRNA and incubated for 24 h. In another set of the experimental group, cells were pre-treated with nimbolide (2.5 µM) for 24 h. Then, cells were stimulated with TNF-α (10 ng/ml) for 30 min except for nimbolide alone (NIM) group (2.5 µM). The expression of TNF-α, NF-κB and HDAC-3 levels were measured by ( c ) western blotting and ( d ) confocal analysis. The images were captured at ×400 magnification

Techniques Used: Expressing, Translocation Assay, Western Blot, Transfection, Incubation

Nimbolide modulates the pro- and anti-inflammatory cytokines and chemokines. The proinflammatory cytokines and chemokines (IL-6, IL-12 (p40), MIP-1α, MIP-1β, and TNF-α) and anti-inflammatory cytokines (IL-4, IL-10, and IL-13) expression were determined by multiplex and ELISA. a – h Animal lung tissue lysate was prepared and evaluated for the levels of aforementioned inflammatory cytokines and chemokines by multiplex. i , j lung tissue supernatants were subjected to ELISA to determine IL-1β and TGF-β cytokines expression. k Griess assay was performed to determine the nitrite levels in cell lysate of lung tissues. l The antioxidant GSH levels were measured in lung tissue. m The MPO as well as ( n ) iNOS, nitrotyrosine, Nrf-2, HO-1, and SOD-1 protein expressions were determined by immunoblot analysis. Data presented as mean ± SEM (n=8 mice per group). * P
Figure Legend Snippet: Nimbolide modulates the pro- and anti-inflammatory cytokines and chemokines. The proinflammatory cytokines and chemokines (IL-6, IL-12 (p40), MIP-1α, MIP-1β, and TNF-α) and anti-inflammatory cytokines (IL-4, IL-10, and IL-13) expression were determined by multiplex and ELISA. a – h Animal lung tissue lysate was prepared and evaluated for the levels of aforementioned inflammatory cytokines and chemokines by multiplex. i , j lung tissue supernatants were subjected to ELISA to determine IL-1β and TGF-β cytokines expression. k Griess assay was performed to determine the nitrite levels in cell lysate of lung tissues. l The antioxidant GSH levels were measured in lung tissue. m The MPO as well as ( n ) iNOS, nitrotyrosine, Nrf-2, HO-1, and SOD-1 protein expressions were determined by immunoblot analysis. Data presented as mean ± SEM (n=8 mice per group). * P

Techniques Used: Expressing, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Griess Assay, Mouse Assay

5) Product Images from "Steppogenin Isolated from Cudrania tricuspidata Shows Antineuroinflammatory Effects via NF-κB and MAPK Pathways in LPS-Stimulated BV2 and Primary Rat Microglial Cells"

Article Title: Steppogenin Isolated from Cudrania tricuspidata Shows Antineuroinflammatory Effects via NF-κB and MAPK Pathways in LPS-Stimulated BV2 and Primary Rat Microglial Cells

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules22122130

The effects of steppogenin ( 1 ) on nitrite ( A ) production and iNOS and COX-2 expression ( B ) in lipopolysaccharide (LPS)-stimulated primary rat microglial cells. ( A , B ) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 24 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensities were quantified by densitometry and normalized to the intensities of the β-actin band; the normalized values are presented below each band. ** p
Figure Legend Snippet: The effects of steppogenin ( 1 ) on nitrite ( A ) production and iNOS and COX-2 expression ( B ) in lipopolysaccharide (LPS)-stimulated primary rat microglial cells. ( A , B ) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 24 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensities were quantified by densitometry and normalized to the intensities of the β-actin band; the normalized values are presented below each band. ** p

Techniques Used: Expressing

The effects of steppogenin ( 1 ) on nitrite ( A ) and prostaglandin E2 (PGE 2 ) ( B ) production and iNOS and COX-2 expression ( C ) in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. ( A – C ) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 24 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensity was quantified by densitometry and normalized to the intensity of the β-actin band; the normalized values are presented below each band. * p
Figure Legend Snippet: The effects of steppogenin ( 1 ) on nitrite ( A ) and prostaglandin E2 (PGE 2 ) ( B ) production and iNOS and COX-2 expression ( C ) in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. ( A – C ) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 24 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensity was quantified by densitometry and normalized to the intensity of the β-actin band; the normalized values are presented below each band. * p

Techniques Used: Expressing

6) Product Images from "Ameliorative Effects of Scopoletin from Crossostephium chinensis against Inflammation Pain and Its Mechanisms in Mice"

Article Title: Ameliorative Effects of Scopoletin from Crossostephium chinensis against Inflammation Pain and Its Mechanisms in Mice

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2012/595603

Inhibition of iNOS and COX-2 protein expression by scopoletin induced by Carr in mice paw edema for 5th hour. Normal control received 0.9% normal saline. Animals treated with scopoletin (1, 5, and 10 mg/kg) and Indo to injection of Carr right hind paws. The right hind paw tissues were taken at the 5 hour. Then the homogenate was centrifuged and tissue suspended were then prepared and subjected to western blotting using an antibody specific for iNOS and COX-2. β -actin was used as an internal control. (a) Representative western blot from two separate experiments was shown. (b) Relative iNOS and COX-2 protein levels were calculated with reference to Carr-injected mouse. Each point represents the average value for three individual animals . ### compared with sample of control group. The data were presented as mean ± S.E.M. for three different experiments performed in triplicate. ** P
Figure Legend Snippet: Inhibition of iNOS and COX-2 protein expression by scopoletin induced by Carr in mice paw edema for 5th hour. Normal control received 0.9% normal saline. Animals treated with scopoletin (1, 5, and 10 mg/kg) and Indo to injection of Carr right hind paws. The right hind paw tissues were taken at the 5 hour. Then the homogenate was centrifuged and tissue suspended were then prepared and subjected to western blotting using an antibody specific for iNOS and COX-2. β -actin was used as an internal control. (a) Representative western blot from two separate experiments was shown. (b) Relative iNOS and COX-2 protein levels were calculated with reference to Carr-injected mouse. Each point represents the average value for three individual animals . ### compared with sample of control group. The data were presented as mean ± S.E.M. for three different experiments performed in triplicate. ** P

Techniques Used: Inhibition, Expressing, Mouse Assay, Injection, Western Blot

7) Product Images from "Ameliorative Effects of Scopoletin from Crossostephium chinensis against Inflammation Pain and Its Mechanisms in Mice"

Article Title: Ameliorative Effects of Scopoletin from Crossostephium chinensis against Inflammation Pain and Its Mechanisms in Mice

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2012/595603

Inhibition of iNOS and COX-2 protein expression by scopoletin induced by Carr in mice paw edema for 5th hour. Normal control received 0.9% normal saline. Animals treated with scopoletin (1, 5, and 10 mg/kg) and Indo to injection of Carr right hind paws. The right hind paw tissues were taken at the 5 hour. Then the homogenate was centrifuged and tissue suspended were then prepared and subjected to western blotting using an antibody specific for iNOS and COX-2.  β -actin was used as an internal control. (a) Representative western blot from two separate experiments was shown. (b) Relative iNOS and COX-2 protein levels were calculated with reference to Carr-injected mouse. Each point represents the  average value  for three  individual animals .  ### compared with sample of control group. The data were presented as mean ± S.E.M. for three different experiments performed in triplicate. ** P
Figure Legend Snippet: Inhibition of iNOS and COX-2 protein expression by scopoletin induced by Carr in mice paw edema for 5th hour. Normal control received 0.9% normal saline. Animals treated with scopoletin (1, 5, and 10 mg/kg) and Indo to injection of Carr right hind paws. The right hind paw tissues were taken at the 5 hour. Then the homogenate was centrifuged and tissue suspended were then prepared and subjected to western blotting using an antibody specific for iNOS and COX-2. β -actin was used as an internal control. (a) Representative western blot from two separate experiments was shown. (b) Relative iNOS and COX-2 protein levels were calculated with reference to Carr-injected mouse. Each point represents the average value for three individual animals . ### compared with sample of control group. The data were presented as mean ± S.E.M. for three different experiments performed in triplicate. ** P

Techniques Used: Inhibition, Expressing, Mouse Assay, Injection, Western Blot

8) Product Images from "Crocin Suppresses LPS-Stimulated Expression of Inducible Nitric Oxide Synthase by Upregulation of Heme Oxygenase-1 via Calcium/Calmodulin-Dependent Protein Kinase 4"

Article Title: Crocin Suppresses LPS-Stimulated Expression of Inducible Nitric Oxide Synthase by Upregulation of Heme Oxygenase-1 via Calcium/Calmodulin-Dependent Protein Kinase 4

Journal: Mediators of Inflammation

doi: 10.1155/2014/728709

CAMK4 is necessary for crocin-mediated inhibition of iNOS expression in LPS-stimulated macrophages. RAW 264.7 cells were transfected with CAMK4 siRNA or control siRNA and then treated with crocin (500 μ M). After 3 h, cells were incubated with LPS (0.1 μ g/mL) for 24 h. Equal amounts of cytosolic extract were analyzed by Western blotting. Tubulin was used as a loading control.
Figure Legend Snippet: CAMK4 is necessary for crocin-mediated inhibition of iNOS expression in LPS-stimulated macrophages. RAW 264.7 cells were transfected with CAMK4 siRNA or control siRNA and then treated with crocin (500 μ M). After 3 h, cells were incubated with LPS (0.1 μ g/mL) for 24 h. Equal amounts of cytosolic extract were analyzed by Western blotting. Tubulin was used as a loading control.

Techniques Used: Inhibition, Expressing, Transfection, Incubation, Western Blot

9) Product Images from "S100a9 Knockdown Decreases the Memory Impairment and the Neuropathology in Tg2576 Mice, AD Animal Model"

Article Title: S100a9 Knockdown Decreases the Memory Impairment and the Neuropathology in Tg2576 Mice, AD Animal Model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0008840

S100a9 induced by Aβ or CT increased [Ca 2+ ] i level and proinflammatory cytokines in BV2 cells. (A) [Ca 2+ ] i images obtained by Fluo3 AM at 48 h after treatment with 10 µM CT and 20 nM si-S100a9 in BV2 cells. Scale bars represent 50 µm. si-CTL is a control containing the scrambled sequence of S100a9 (B) The histogram shows the ratio of [Ca 2+ ] i levels to NC group. [Ca 2+ ] i levels in BV2 cells treated with CT and si-S100a9 was compared to that in BV2 cells with CT and si-CTL (control) (n = 5). (C) After treatment with 1, 5, or 10 µM CT and 5 or 10 µM Aβ to BV2 cells, the levels of the pro-inflammatory cytokines, IL-1β and TNF-α, were measured by western blotting. The membrane was stripped and reprobed with GAPDH to confirm equal loading and with 6E10 antibody to confirm intracellular CT and Aβ. (D) At 48 h post-treatment with si-S100a9, expressions of S100a9, IL-1β, TNF-α and iNOS were attenuated. The membrane was stripped and reprobed with GAPDH to confirm equal loading. (E) At 48 h post-treatment with 10 µM CT and si-S100a9, NO release (µM) was measured by Griess reagent. LPS (1 µg/ml) was used as a positive control. All data are presented as the means ± SEM (n = 10). NC: negative control (non-treated cell) **P
Figure Legend Snippet: S100a9 induced by Aβ or CT increased [Ca 2+ ] i level and proinflammatory cytokines in BV2 cells. (A) [Ca 2+ ] i images obtained by Fluo3 AM at 48 h after treatment with 10 µM CT and 20 nM si-S100a9 in BV2 cells. Scale bars represent 50 µm. si-CTL is a control containing the scrambled sequence of S100a9 (B) The histogram shows the ratio of [Ca 2+ ] i levels to NC group. [Ca 2+ ] i levels in BV2 cells treated with CT and si-S100a9 was compared to that in BV2 cells with CT and si-CTL (control) (n = 5). (C) After treatment with 1, 5, or 10 µM CT and 5 or 10 µM Aβ to BV2 cells, the levels of the pro-inflammatory cytokines, IL-1β and TNF-α, were measured by western blotting. The membrane was stripped and reprobed with GAPDH to confirm equal loading and with 6E10 antibody to confirm intracellular CT and Aβ. (D) At 48 h post-treatment with si-S100a9, expressions of S100a9, IL-1β, TNF-α and iNOS were attenuated. The membrane was stripped and reprobed with GAPDH to confirm equal loading. (E) At 48 h post-treatment with 10 µM CT and si-S100a9, NO release (µM) was measured by Griess reagent. LPS (1 µg/ml) was used as a positive control. All data are presented as the means ± SEM (n = 10). NC: negative control (non-treated cell) **P

Techniques Used: CTL Assay, Sequencing, Western Blot, Positive Control, Negative Control

10) Product Images from "SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI)"

Article Title: SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI)

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-9-237

Expression of PDI and SOD1 in cultured astrocytes following OGD / reperfusion. A . PDI expression in cultured astrocytes showed an increasing trend in the process of reperfusion and peaked at OGD 8 h/reperfusion 24 h. B . SOD1 expression in cultured astrocytes increased gradually during the process of OGD/reperfusion and reached a peak at OGD 8 h/reperfusion 24 h. C . The anti-SOD1 antibody co-precipitated PDI, and the anti-PDI antibody co-precipitated SOD1 in cultured astrocytes. Three separate immunoprecipitations were performed, and data were analyzed in triplicate. Data were presented as mean ± SEM; *, P
Figure Legend Snippet: Expression of PDI and SOD1 in cultured astrocytes following OGD / reperfusion. A . PDI expression in cultured astrocytes showed an increasing trend in the process of reperfusion and peaked at OGD 8 h/reperfusion 24 h. B . SOD1 expression in cultured astrocytes increased gradually during the process of OGD/reperfusion and reached a peak at OGD 8 h/reperfusion 24 h. C . The anti-SOD1 antibody co-precipitated PDI, and the anti-PDI antibody co-precipitated SOD1 in cultured astrocytes. Three separate immunoprecipitations were performed, and data were analyzed in triplicate. Data were presented as mean ± SEM; *, P

Techniques Used: Expressing, Cell Culture

Distribution of ubiquitin - conjugated proteins and SOD1 in cultured astrocytes following OGD / reperfusion. A . In astrocytes, the ubiquitin immunoreactivity in the normoxic control group was evenly distributed in cytoplasm and nucleus. Following OGD 8 h/reperfusion 16 h, the diffused distribution of free ubiquitin lost nuclear staining and clustered immunoreactivity appeared near the nucleus. 1400W attenuated the punctuated ubiquitin perinuclear distribution B . Colocalization of ubiquitin and SOD1. Following OGD 8 h/reperfusion 16 h, were identified. SOD1 immunoreactivity colocalized with ubiquitinated-protein aggregates and showed a pattern of punctuated perinuclear-nuclear localization. Scale bar, 100 μm; green, ubiquitin immunosignals; red, SOD1 immunosignals; blue, nucleus staining with Hoechst 33342.
Figure Legend Snippet: Distribution of ubiquitin - conjugated proteins and SOD1 in cultured astrocytes following OGD / reperfusion. A . In astrocytes, the ubiquitin immunoreactivity in the normoxic control group was evenly distributed in cytoplasm and nucleus. Following OGD 8 h/reperfusion 16 h, the diffused distribution of free ubiquitin lost nuclear staining and clustered immunoreactivity appeared near the nucleus. 1400W attenuated the punctuated ubiquitin perinuclear distribution B . Colocalization of ubiquitin and SOD1. Following OGD 8 h/reperfusion 16 h, were identified. SOD1 immunoreactivity colocalized with ubiquitinated-protein aggregates and showed a pattern of punctuated perinuclear-nuclear localization. Scale bar, 100 μm; green, ubiquitin immunosignals; red, SOD1 immunosignals; blue, nucleus staining with Hoechst 33342.

Techniques Used: Cell Culture, Staining

11) Product Images from "In vitro and in vivo anti-cancer activities of Kuding tea (Ilex kudingcha C.J. Tseng) against oral cancer"

Article Title: In vitro and in vivo anti-cancer activities of Kuding tea (Ilex kudingcha C.J. Tseng) against oral cancer

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2013.1450

Effects of various concentrations of Kuding tea on the mRNA (left) and protein (right) expression levels of MMPs and TIMPs in human tongue carcinoma TCA8113 cells.
Figure Legend Snippet: Effects of various concentrations of Kuding tea on the mRNA (left) and protein (right) expression levels of MMPs and TIMPs in human tongue carcinoma TCA8113 cells.

Techniques Used: Expressing

12) Product Images from "Foxo1 Links Hyperglycemia to LDL Oxidation and Endothelial Nitric Oxide Synthase Dysfunction in Vascular Endothelial Cells"

Article Title: Foxo1 Links Hyperglycemia to LDL Oxidation and Endothelial Nitric Oxide Synthase Dysfunction in Vascular Endothelial Cells

Journal: Diabetes

doi: 10.2337/db09-0167

iNOS-dependent NO and ROS/peroxynitrite generation in response to high glucose or oxidative stress in HAECs and in mice. A–F : HAECs were incubated in medium containing 5.5 mmol/l glucose (–), 25 mmol/l glucose (HG) or 25 mmol/l mannitol (Man) for 48 h, or H 2 O 2 (0.15 or 0.5 mmol/l) for 12 h with ( E and F ) or without ( A–D ) either iNOS (1400W) or eNOS inhibitors ( l -NAME). A , C , and F : Representative images ( upper panel s) and calculated relative intensities ( lower panel s) of NO production using DAF2-DA. B : ROS/peroxynitrite production using carboxy-H 2 DCFDA. D and E : iNOS and eNOS protein ( upper panel ) and mRNA ( lower panel ) expression. G : Blood glucose, ( H ) aortic iNOS mRNA expression, ( I ) iNOS immunohistochemistry in aortic sections from C57BL/6J mice (first and second panel from the left) and from Tie2-cre/ Foxo1 flox/flox and Foxo1 flox/flox mice (third and fourth panel from the left), and ( J ) plasma lipid peroxide levels (TBARS) in STZ-induced diabetic and saline-treated control mice ( n = 6 for each group). The data were obtained two weeks after STZ injection. * P
Figure Legend Snippet: iNOS-dependent NO and ROS/peroxynitrite generation in response to high glucose or oxidative stress in HAECs and in mice. A–F : HAECs were incubated in medium containing 5.5 mmol/l glucose (–), 25 mmol/l glucose (HG) or 25 mmol/l mannitol (Man) for 48 h, or H 2 O 2 (0.15 or 0.5 mmol/l) for 12 h with ( E and F ) or without ( A–D ) either iNOS (1400W) or eNOS inhibitors ( l -NAME). A , C , and F : Representative images ( upper panel s) and calculated relative intensities ( lower panel s) of NO production using DAF2-DA. B : ROS/peroxynitrite production using carboxy-H 2 DCFDA. D and E : iNOS and eNOS protein ( upper panel ) and mRNA ( lower panel ) expression. G : Blood glucose, ( H ) aortic iNOS mRNA expression, ( I ) iNOS immunohistochemistry in aortic sections from C57BL/6J mice (first and second panel from the left) and from Tie2-cre/ Foxo1 flox/flox and Foxo1 flox/flox mice (third and fourth panel from the left), and ( J ) plasma lipid peroxide levels (TBARS) in STZ-induced diabetic and saline-treated control mice ( n = 6 for each group). The data were obtained two weeks after STZ injection. * P

Techniques Used: Mouse Assay, Incubation, Expressing, Immunohistochemistry, Injection

Foxo1ADA increases iNOS mRNA, NO, and ROS/peroxynitrite generation. HAECs were transduced with increasing concentrations of HA-Foxo1ADA for 24 h ( A–D and F ) with or without pretreatment of the eNOS inhibitor, l -NAME, or iNOS inhibitor, 1400W ( E and G ). A : Endogenous and exogenous Foxo1 Western blotting using anti-Foxo1 and anti-HA antibodies. B and E : NO production using DAF-2DA. C : ROS/peroxynitrite production using carboxy-H 2 DCFDA. D : iNOS and eNOS proteins ( upper panel ) and mRNA ( lower panel ) expression levels. F and G : Total amount of NOx concentration in the medium. * P
Figure Legend Snippet: Foxo1ADA increases iNOS mRNA, NO, and ROS/peroxynitrite generation. HAECs were transduced with increasing concentrations of HA-Foxo1ADA for 24 h ( A–D and F ) with or without pretreatment of the eNOS inhibitor, l -NAME, or iNOS inhibitor, 1400W ( E and G ). A : Endogenous and exogenous Foxo1 Western blotting using anti-Foxo1 and anti-HA antibodies. B and E : NO production using DAF-2DA. C : ROS/peroxynitrite production using carboxy-H 2 DCFDA. D : iNOS and eNOS proteins ( upper panel ) and mRNA ( lower panel ) expression levels. F and G : Total amount of NOx concentration in the medium. * P

Techniques Used: Transduction, Western Blot, Expressing, Concentration Assay

13) Product Images from "Aberrant fetal macrophage/microglial reactions to cytomegalovirus infection"

Article Title: Aberrant fetal macrophage/microglial reactions to cytomegalovirus infection

Journal: Annals of Clinical and Translational Neurology

doi: 10.1002/acn3.88

MCMV infection impairs cerebral corticogenesis in the upper layer rather than the lower layer. Detection of Tbr1 (green) and Brn2 (red), as lower and upper layer markers, respectively, by immunofluorescence staining of sections of MCMV-infected fetal cerebra at 5 dpi. Nuclei were stained with DAPI (blue). (A) The localization of Tbr1 + or Brn2 + cells in a representative cerebral section of E18.5 fetus infected with mock virus (upper panel) or MCMV (lower panel). Despite no differences in the number of Tbr1 + neurons in the lower layer between the mock- and MCMV-infected cerebra, the number of Brn2 + neurons in the upper layer in the MCMV-infected cerebrum decreased. Scale bar = 100 μ m. (B) The numbers of Tbr1 + and Brn2 + neurons in the upper and lower layers in a 1.6 mm 2 area of MCMV-infected cerebrum and in the corresponding area of mock-infected cerebrum. Data are expressed as mean ± SEM of three fetuses. * P
Figure Legend Snippet: MCMV infection impairs cerebral corticogenesis in the upper layer rather than the lower layer. Detection of Tbr1 (green) and Brn2 (red), as lower and upper layer markers, respectively, by immunofluorescence staining of sections of MCMV-infected fetal cerebra at 5 dpi. Nuclei were stained with DAPI (blue). (A) The localization of Tbr1 + or Brn2 + cells in a representative cerebral section of E18.5 fetus infected with mock virus (upper panel) or MCMV (lower panel). Despite no differences in the number of Tbr1 + neurons in the lower layer between the mock- and MCMV-infected cerebra, the number of Brn2 + neurons in the upper layer in the MCMV-infected cerebrum decreased. Scale bar = 100 μ m. (B) The numbers of Tbr1 + and Brn2 + neurons in the upper and lower layers in a 1.6 mm 2 area of MCMV-infected cerebrum and in the corresponding area of mock-infected cerebrum. Data are expressed as mean ± SEM of three fetuses. * P

Techniques Used: Infection, Immunofluorescence, Staining

14) Product Images from "miR-155 Deletion in Mice Overcomes Neuron-Intrinsic and Neuron-Extrinsic Barriers to Spinal Cord Repair"

Article Title: miR-155 Deletion in Mice Overcomes Neuron-Intrinsic and Neuron-Extrinsic Barriers to Spinal Cord Repair

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0735-16.2016

miR-155 is required for macrophages to acquire an inflammatory phenotype. A , BMDMs stimulated with IFN-γ + LPS increase their expression of miR-155 by ∼3000%. miR-155 expression was not increased by IL-4. B , C , WT and miR-155 KO macrophages were stimulated with media, IFN-γ + LPS, or IL-4 for 24 h (loading control: α-tubulin) and then cell lysates were collected to assess expression of canonical M1 (iNOS) and M2 (Ym1, Arg1, CD206) markers. Inset in C shows that, consistent with a significant reduction in iNOS, nitric oxide (NO) release was reduced (∼30%) in miR-155 KO macrophages after stimulation with IFN-γ + LPS. miR-155 KO macrophages also expressed higher levels of Ym1 (* p
Figure Legend Snippet: miR-155 is required for macrophages to acquire an inflammatory phenotype. A , BMDMs stimulated with IFN-γ + LPS increase their expression of miR-155 by ∼3000%. miR-155 expression was not increased by IL-4. B , C , WT and miR-155 KO macrophages were stimulated with media, IFN-γ + LPS, or IL-4 for 24 h (loading control: α-tubulin) and then cell lysates were collected to assess expression of canonical M1 (iNOS) and M2 (Ym1, Arg1, CD206) markers. Inset in C shows that, consistent with a significant reduction in iNOS, nitric oxide (NO) release was reduced (∼30%) in miR-155 KO macrophages after stimulation with IFN-γ + LPS. miR-155 KO macrophages also expressed higher levels of Ym1 (* p

Techniques Used: Expressing

15) Product Images from "Inhibition of ROS-Activated p38MAPK Pathway is Involved in the Protective Effect of H2S Against Chemical Hypoxia-Induced Inflammation in PC12 Cells"

Article Title: Inhibition of ROS-Activated p38MAPK Pathway is Involved in the Protective Effect of H2S Against Chemical Hypoxia-Induced Inflammation in PC12 Cells

Journal: Neurochemical Research

doi: 10.1007/s11064-013-1044-x

NaHS attenuates CoCl 2 -induced expression levels of iNOS and nNOS as well as NO production in PC12 cells. PC12 cells were treated with 600 μmol/l CoCl 2 for 24 h in the absence or presence of the preconditioning with 400 μmol/l NaHS for 30 min. The expression levels of iNOS ( a ) and nNOS ( c ) were determined by Western blot assay. b and d Densitometric analysis of the results from a and c , respectively. e PC12 cells were treated with 600 μmol/l CoCl 2 for 48 h in the presence or absence of pretreatment with 400 μmol/l NaHS for 30 min. Nitrite in the culture supernatant was determined using the Griess reagent as described in “Materials and Methods”. Data were presented as the mean ± SEM (n = 5). ## P
Figure Legend Snippet: NaHS attenuates CoCl 2 -induced expression levels of iNOS and nNOS as well as NO production in PC12 cells. PC12 cells were treated with 600 μmol/l CoCl 2 for 24 h in the absence or presence of the preconditioning with 400 μmol/l NaHS for 30 min. The expression levels of iNOS ( a ) and nNOS ( c ) were determined by Western blot assay. b and d Densitometric analysis of the results from a and c , respectively. e PC12 cells were treated with 600 μmol/l CoCl 2 for 48 h in the presence or absence of pretreatment with 400 μmol/l NaHS for 30 min. Nitrite in the culture supernatant was determined using the Griess reagent as described in “Materials and Methods”. Data were presented as the mean ± SEM (n = 5). ## P

Techniques Used: Expressing, Western Blot

NaHS, L-canavanine or 7-nitroindazole inhibits CoCl 2 -induced cytotoxicity in PC12 cells. a PC12 cells were pretreated with L-canavanine, an inhibitor of iNOS, at indicated concentrations for 60 min before exposure to 600 μmol/l CoCl 2 for 24 h. b PC12 cells were pretreated with 400 μmol/l NaHS for 30 min or 5 μmol/l L-canavanine for 60 min or 250 μmol/l 7-nitroindazole (7-NI), an inhibitor of nNOS, for 30 min prior to exposure to 600 μmol/l CoCl 2 for 24 h. Cell viability was measured by the CCK-8 assay. Data were presented as mean ± SEM (n = 3). ## P
Figure Legend Snippet: NaHS, L-canavanine or 7-nitroindazole inhibits CoCl 2 -induced cytotoxicity in PC12 cells. a PC12 cells were pretreated with L-canavanine, an inhibitor of iNOS, at indicated concentrations for 60 min before exposure to 600 μmol/l CoCl 2 for 24 h. b PC12 cells were pretreated with 400 μmol/l NaHS for 30 min or 5 μmol/l L-canavanine for 60 min or 250 μmol/l 7-nitroindazole (7-NI), an inhibitor of nNOS, for 30 min prior to exposure to 600 μmol/l CoCl 2 for 24 h. Cell viability was measured by the CCK-8 assay. Data were presented as mean ± SEM (n = 3). ## P

Techniques Used: CCK-8 Assay

16) Product Images from "Energy restriction, exercise and atorvastatin treatment improve endothelial dysfunction and inhibit miRNA-155 in the erectile tissue of the aged rat."

Article Title: Energy restriction, exercise and atorvastatin treatment improve endothelial dysfunction and inhibit miRNA-155 in the erectile tissue of the aged rat.

Journal: Nutrition & Metabolism

doi: 10.1186/s12986-018-0265-z

Dual immunolabeling of SIRT1/SIRT7 ( a-e ) and SIRT2/α-actin ( f-j ) in erectile tissue of rats from all experimental groups ( n = 3/group). SIRT1 was identified in the nucleus and cytoplasm (green) mainly in the SMC of all the analyzed tissues ( a-e ) and SIRT7 is apparently more expressed in the endothelium (red) ( a-e ). No marked differences were seen among groups for SIRT1 and SIRT7 expression. SIRT2 expression was observed in cytosol mainly in SMC in all experimental groups, often co-localizing with α-actin labeling ( f-j ). The co-localization seems to be more intense in CC from rats of HF/ER/S/Ex group ( j ). C-control; HF-high-fat diet treated rats; HF/ER-high-fat diet treated rats under energy restriction for 6 months; HF/ER/S-high-fat diet treated rats under energy restriction and atorvastatin treatment for 6 months; HF/ER/S/Ex-high-fat diet treated rats under energy restriction, atorvastatin treatment and exercise for 6 months. VS- vascular space
Figure Legend Snippet: Dual immunolabeling of SIRT1/SIRT7 ( a-e ) and SIRT2/α-actin ( f-j ) in erectile tissue of rats from all experimental groups ( n = 3/group). SIRT1 was identified in the nucleus and cytoplasm (green) mainly in the SMC of all the analyzed tissues ( a-e ) and SIRT7 is apparently more expressed in the endothelium (red) ( a-e ). No marked differences were seen among groups for SIRT1 and SIRT7 expression. SIRT2 expression was observed in cytosol mainly in SMC in all experimental groups, often co-localizing with α-actin labeling ( f-j ). The co-localization seems to be more intense in CC from rats of HF/ER/S/Ex group ( j ). C-control; HF-high-fat diet treated rats; HF/ER-high-fat diet treated rats under energy restriction for 6 months; HF/ER/S-high-fat diet treated rats under energy restriction and atorvastatin treatment for 6 months; HF/ER/S/Ex-high-fat diet treated rats under energy restriction, atorvastatin treatment and exercise for 6 months. VS- vascular space

Techniques Used: Immunolabeling, Expressing, Labeling

17) Product Images from "Interleukin-17-dependent CXCL-13 mediates mucosal vaccine-induced immunity against tuberculosis"

Article Title: Interleukin-17-dependent CXCL-13 mediates mucosal vaccine-induced immunity against tuberculosis

Journal: Mucosal immunology

doi: 10.1038/mi.2012.135

Absence of IL-17 results in impaired lung macrophage activation during vaccine-induced immunity B6, and Il17 −/− were mucosally vaccinated with ESAT6 1–20 in combination with LT-IIb, rested, challenged with Mtb as described in Figure 1 . Formalin-fixed, paraffin embedded lung sections from day 30 Mtb -challenged lungs were analyzed by immunofluorescence for T cell perivascular cuffing (CD3 + ) and quantified using the morphometric tool of the Zeiss Axioplan microscope and a representative image of typical T cell perivascular cuffing shown (a). The number of CD3+ T cells within the granuloma were quantitated as described under method (b). The mean fluorescent intensity (MFI) of MHC Class II I-A b expression on lung macrophages was determined by flow cytometry on day 15 from mucosally vaccinated Mtb -infected mice (Vacc), unvaccinated Mtb -infected mice (Un) or uninfected mice (UI) (c). The number of F4/80 macrophages producing iNOS were enumerated on day 30 post infection in formalin fixed lungs by immunofluorescence and a representative picture shown (d). The data points represent the mean (±SD) of values from 4–6 mice (a–d). *, p ≤0.05, ***p≤0.0005. One experiment representative of two is shown. ns-not significant.
Figure Legend Snippet: Absence of IL-17 results in impaired lung macrophage activation during vaccine-induced immunity B6, and Il17 −/− were mucosally vaccinated with ESAT6 1–20 in combination with LT-IIb, rested, challenged with Mtb as described in Figure 1 . Formalin-fixed, paraffin embedded lung sections from day 30 Mtb -challenged lungs were analyzed by immunofluorescence for T cell perivascular cuffing (CD3 + ) and quantified using the morphometric tool of the Zeiss Axioplan microscope and a representative image of typical T cell perivascular cuffing shown (a). The number of CD3+ T cells within the granuloma were quantitated as described under method (b). The mean fluorescent intensity (MFI) of MHC Class II I-A b expression on lung macrophages was determined by flow cytometry on day 15 from mucosally vaccinated Mtb -infected mice (Vacc), unvaccinated Mtb -infected mice (Un) or uninfected mice (UI) (c). The number of F4/80 macrophages producing iNOS were enumerated on day 30 post infection in formalin fixed lungs by immunofluorescence and a representative picture shown (d). The data points represent the mean (±SD) of values from 4–6 mice (a–d). *, p ≤0.05, ***p≤0.0005. One experiment representative of two is shown. ns-not significant.

Techniques Used: Activation Assay, Formalin-fixed Paraffin-Embedded, Immunofluorescence, Microscopy, Expressing, Flow Cytometry, Cytometry, Infection, Mouse Assay

IL-17 improves the protective efficacy of mucosal vaccination following Mtb challenge B6 mice were mucosally vaccinated with ESAT6 1–20 in LT-IIb, rested and challenged with Mtb as described in Figure 1 . Vaccinated Mtb -challenged mice either received PBS, rIL-17 or rIFNγ (1.5 µg per mouse) intratracheally from day 5 to day 17 post-infection during recall response and the lung bacterial burden was determined on day 30 post vaccination (a). On day 30 post-infection, formalin-fixed, paraffin embedded lung sections were stained with H E or CD3 (red), and B220 (green). Area occupied by inflammatory lesions/lung lobe (b), B cell lymphoid follicles (c) was quantified using the morphometric tool of the Zeiss Axioplan microscope. The representative figure showing a typical inflammatory lesions (d-top panel), B cell follicle (d-middle panel) and CXCL13 expression within B cell follicles (d-bottom panel) is included. The average area of perivascular cuffing from the above mentioned groups were quantified (e). One group of mucosally vaccinated B6 mice received control adenovirus expressing luciferase vector (Vacc+Vector) while a second group received adenovirus overexpressing IL-23 (Vacc+Ad IL-23) (5×10 8 pfu) on the day of vaccination. Mucosally vaccinated and unvaccinated mice were rested and challenged with Mtb and lung bacterial burden was determined on day 30 post infection (f). B6 mice were either subcutaneously vaccinated with 1×10 6 M.bovis BCG, mucosally vaccinated, or subcutaneously vaccinated with M.bovis BCG followed by a period of rest for 30 days and boosted mucosally as described under methods. All groups of mice were then rested for 30 days and challenged with Mtb as described in Figure 1 and lung bacterial burden was determined on day 30 post infection (g). Original magnification for H E sections, 100X; immunofluorescent sections, 200X. The data points represent the mean (±SD) of values from 4–6 mice (a–g). *p≤0.05, **≤0.005, ***p≤0.0005. One experiment representative of two is shown. ns-not significant.
Figure Legend Snippet: IL-17 improves the protective efficacy of mucosal vaccination following Mtb challenge B6 mice were mucosally vaccinated with ESAT6 1–20 in LT-IIb, rested and challenged with Mtb as described in Figure 1 . Vaccinated Mtb -challenged mice either received PBS, rIL-17 or rIFNγ (1.5 µg per mouse) intratracheally from day 5 to day 17 post-infection during recall response and the lung bacterial burden was determined on day 30 post vaccination (a). On day 30 post-infection, formalin-fixed, paraffin embedded lung sections were stained with H E or CD3 (red), and B220 (green). Area occupied by inflammatory lesions/lung lobe (b), B cell lymphoid follicles (c) was quantified using the morphometric tool of the Zeiss Axioplan microscope. The representative figure showing a typical inflammatory lesions (d-top panel), B cell follicle (d-middle panel) and CXCL13 expression within B cell follicles (d-bottom panel) is included. The average area of perivascular cuffing from the above mentioned groups were quantified (e). One group of mucosally vaccinated B6 mice received control adenovirus expressing luciferase vector (Vacc+Vector) while a second group received adenovirus overexpressing IL-23 (Vacc+Ad IL-23) (5×10 8 pfu) on the day of vaccination. Mucosally vaccinated and unvaccinated mice were rested and challenged with Mtb and lung bacterial burden was determined on day 30 post infection (f). B6 mice were either subcutaneously vaccinated with 1×10 6 M.bovis BCG, mucosally vaccinated, or subcutaneously vaccinated with M.bovis BCG followed by a period of rest for 30 days and boosted mucosally as described under methods. All groups of mice were then rested for 30 days and challenged with Mtb as described in Figure 1 and lung bacterial burden was determined on day 30 post infection (g). Original magnification for H E sections, 100X; immunofluorescent sections, 200X. The data points represent the mean (±SD) of values from 4–6 mice (a–g). *p≤0.05, **≤0.005, ***p≤0.0005. One experiment representative of two is shown. ns-not significant.

Techniques Used: Mouse Assay, Infection, Formalin-fixed Paraffin-Embedded, Staining, Microscopy, Expressing, Luciferase, Plasmid Preparation

IL-17, but not IFNγ is crucial for lymphocytic infiltration, B cell lymphoid follicle and granuloma formation in lungs of mucosally vaccinated Mtb -challenged mice B6, Ifng −/− and Il17 −/− were mucosally vaccinated with ESAT6 1–20 in combination with LT-IIb (Vacc) and rested for 30 days, then challenged with ~100 CFU Mtb by the aerosol route. On day 30 post challenge, formalin fixed lung samples were stained with H E or CD3 (red) B220 (green) and the area occupied by granuloma as inflammatory lesions (a) and B cell lymphoid follicles (b) quantified using the morphometric tool of the Zeiss Axioplan microscope. Representative pictures of inflammatory lesions (c-top panel) and B cell lymphoid follicles (c-bottom panel) are shown. Original magnification for H E sections, 100X, B cell follicles, 200X. Ifng −/− mice were vaccinated, rested, Mtb -infected and treated with isotype control antibody or treated with IL-17 neutralizing antibody between day 5 and 21 (100 µg/mouse every 48 hours), and sacrificed on day 30 post infection. Formalin fixed samples were stained with H E or CD3 (red) B220 (green) and the area occupied by inflammatory lesions /lung lobe (d) and average size of B cell lymphoid follicles harboring CD3 + lymphocytes (e) was quantified by using the morphometric tool of the Zeiss Axioplan microscope. Representative figures showing inflammatory lesions (f-top panel) and B cell lymphoid follicles (f-bottom panel) are shown. The data points represent the mean (±SD) of values from 4–6 mice (a–f). **≤0.005, ***p≤0.0005. One experiment representative of two is shown.
Figure Legend Snippet: IL-17, but not IFNγ is crucial for lymphocytic infiltration, B cell lymphoid follicle and granuloma formation in lungs of mucosally vaccinated Mtb -challenged mice B6, Ifng −/− and Il17 −/− were mucosally vaccinated with ESAT6 1–20 in combination with LT-IIb (Vacc) and rested for 30 days, then challenged with ~100 CFU Mtb by the aerosol route. On day 30 post challenge, formalin fixed lung samples were stained with H E or CD3 (red) B220 (green) and the area occupied by granuloma as inflammatory lesions (a) and B cell lymphoid follicles (b) quantified using the morphometric tool of the Zeiss Axioplan microscope. Representative pictures of inflammatory lesions (c-top panel) and B cell lymphoid follicles (c-bottom panel) are shown. Original magnification for H E sections, 100X, B cell follicles, 200X. Ifng −/− mice were vaccinated, rested, Mtb -infected and treated with isotype control antibody or treated with IL-17 neutralizing antibody between day 5 and 21 (100 µg/mouse every 48 hours), and sacrificed on day 30 post infection. Formalin fixed samples were stained with H E or CD3 (red) B220 (green) and the area occupied by inflammatory lesions /lung lobe (d) and average size of B cell lymphoid follicles harboring CD3 + lymphocytes (e) was quantified by using the morphometric tool of the Zeiss Axioplan microscope. Representative figures showing inflammatory lesions (f-top panel) and B cell lymphoid follicles (f-bottom panel) are shown. The data points represent the mean (±SD) of values from 4–6 mice (a–f). **≤0.005, ***p≤0.0005. One experiment representative of two is shown.

Techniques Used: Mouse Assay, Staining, Microscopy, Infection

CXCL13 is required for mucosal vaccine-induced immunity against TB B6 and Cxcl13 −/− mice were vaccinated with ESAT6 1–20 in Lt-IIb, rested for 30 days and challenged with Mtb as described in Figure 1 and lung bacterial burden was determined (a). (n=8–10 mice combined over two separate experiments). B6 and Cxcl13 −/− mice were mucosally vaccinated, and number of ESAT6 1–20 -specific IL-17 producing CD4 + T cells in the lungs were determined by ELISpot on day 15 post vaccination (b). Area occupied by inflammatory lesions/lung lobe (c) and average size of B cell lymphoid follicles (d) was quantified using the morphometric tool of the Zeiss Axioplan microscope in formalin-fixed day 30 Mtb -challenged lungs. Lung sections from B6 and Cxcl13 −/− mucosally vaccinated Mtb -infected mice were stained with H E (e-top panel) or CD3 (red), and B220 (green) (e-bottom panel) on day 30 post infection. Arrows point to perivascular cuffs in Cxcl13 −/− mice. Formalin-fixed, paraffin embedded lung sections were analyzed by immunofluorescence for T cell perivascular cuffing (CD3 + staining) (f), number of CD3+ T cells that localized within the granuloma (g) and number of iNOS + cells per inflammatory lesions (h). T cell perivascular cuffing was quantified in formalin fixed lung sections (CD3 + ) using the morphometric tool of the Zeiss Axioplan microscope (f). CD3+ T cells and F4/80 expressing macrophages producing INOS were enumerated by immunofluorescence and representative pictures are shown (g,h). The data points represent the mean (±SD) of values from 4–6 mice (b–h). **≤0.005, ***p≤0.0005. One experiment representative of two is shown. ns-not significant.
Figure Legend Snippet: CXCL13 is required for mucosal vaccine-induced immunity against TB B6 and Cxcl13 −/− mice were vaccinated with ESAT6 1–20 in Lt-IIb, rested for 30 days and challenged with Mtb as described in Figure 1 and lung bacterial burden was determined (a). (n=8–10 mice combined over two separate experiments). B6 and Cxcl13 −/− mice were mucosally vaccinated, and number of ESAT6 1–20 -specific IL-17 producing CD4 + T cells in the lungs were determined by ELISpot on day 15 post vaccination (b). Area occupied by inflammatory lesions/lung lobe (c) and average size of B cell lymphoid follicles (d) was quantified using the morphometric tool of the Zeiss Axioplan microscope in formalin-fixed day 30 Mtb -challenged lungs. Lung sections from B6 and Cxcl13 −/− mucosally vaccinated Mtb -infected mice were stained with H E (e-top panel) or CD3 (red), and B220 (green) (e-bottom panel) on day 30 post infection. Arrows point to perivascular cuffs in Cxcl13 −/− mice. Formalin-fixed, paraffin embedded lung sections were analyzed by immunofluorescence for T cell perivascular cuffing (CD3 + staining) (f), number of CD3+ T cells that localized within the granuloma (g) and number of iNOS + cells per inflammatory lesions (h). T cell perivascular cuffing was quantified in formalin fixed lung sections (CD3 + ) using the morphometric tool of the Zeiss Axioplan microscope (f). CD3+ T cells and F4/80 expressing macrophages producing INOS were enumerated by immunofluorescence and representative pictures are shown (g,h). The data points represent the mean (±SD) of values from 4–6 mice (b–h). **≤0.005, ***p≤0.0005. One experiment representative of two is shown. ns-not significant.

Techniques Used: Mouse Assay, Enzyme-linked Immunospot, Microscopy, Infection, Staining, Formalin-fixed Paraffin-Embedded, Immunofluorescence, Expressing

Mucosal vaccination induces vaccine-induced protection and robust Th17 responses following Mtb challenge B6 mice were vaccinated mucosally vaccinated and boosted with ESAT6 1–20 in combination with LT-IIb (Vacc-ESAT6) or sham vaccinated (Vacc-Sham) via the intranasal route. Control unvaccinated mice were also included (Un). Mucosally vaccinated B6 mice were rested for 30 (a) days or 100 days (b), infected with aerosolized Mtb (100 cfu) and lung bacterial burden was determined on day 30 post infection. On day 30 post infection, lungs were fixed in 10% formalin, embedded in paraffin and inflammatory lesions and lymphoid structure formation were assessed in formalin-fixed lungs by staining with H E (c-left panel); or CD3 (red) and B220 (green) (c-right panel). Original magnification for H E sections, 100X and immunofluorescence staining, 200X. B6 mice were mucosally vaccinated as described before and the number of cytokine-producing ESAT6 1–20 -specific CD4 + T cells in the lungs were determined by ELISpot assay on day 14 (d) or day 100 post-vaccination (e) or in day 15 post Mtb -challenge (f) by antigen-driven ELISpot assay. The Log10 fold induction of IFNγ and IL-17 mRNA was determined in cells isolated from mucosally vaccinated Mtb -challenged lungs when compared to levels expressed in cell isolated from unvaccinated Mtb -challenged lungs by RT-PCR (g). The data points represent the mean (±SD) of values from 4–6 mice (a–g). **≤0.005, ***p≤0.0005. One experiment representative of two is shown.
Figure Legend Snippet: Mucosal vaccination induces vaccine-induced protection and robust Th17 responses following Mtb challenge B6 mice were vaccinated mucosally vaccinated and boosted with ESAT6 1–20 in combination with LT-IIb (Vacc-ESAT6) or sham vaccinated (Vacc-Sham) via the intranasal route. Control unvaccinated mice were also included (Un). Mucosally vaccinated B6 mice were rested for 30 (a) days or 100 days (b), infected with aerosolized Mtb (100 cfu) and lung bacterial burden was determined on day 30 post infection. On day 30 post infection, lungs were fixed in 10% formalin, embedded in paraffin and inflammatory lesions and lymphoid structure formation were assessed in formalin-fixed lungs by staining with H E (c-left panel); or CD3 (red) and B220 (green) (c-right panel). Original magnification for H E sections, 100X and immunofluorescence staining, 200X. B6 mice were mucosally vaccinated as described before and the number of cytokine-producing ESAT6 1–20 -specific CD4 + T cells in the lungs were determined by ELISpot assay on day 14 (d) or day 100 post-vaccination (e) or in day 15 post Mtb -challenge (f) by antigen-driven ELISpot assay. The Log10 fold induction of IFNγ and IL-17 mRNA was determined in cells isolated from mucosally vaccinated Mtb -challenged lungs when compared to levels expressed in cell isolated from unvaccinated Mtb -challenged lungs by RT-PCR (g). The data points represent the mean (±SD) of values from 4–6 mice (a–g). **≤0.005, ***p≤0.0005. One experiment representative of two is shown.

Techniques Used: Mouse Assay, Infection, Staining, Immunofluorescence, Enzyme-linked Immunospot, Isolation, Reverse Transcription Polymerase Chain Reaction

18) Product Images from "Dietary supplementation with cholesterol and docosahexaenoic acid increases the activity of the arginine-nitric oxide pathway in tissues of young pigs"

Article Title: Dietary supplementation with cholesterol and docosahexaenoic acid increases the activity of the arginine-nitric oxide pathway in tissues of young pigs

Journal: Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society

doi: 10.1016/j.niox.2008.05.002

Total nNOS (A), phosphorylated nNOS at Ser 852 (B), total eNOS (C), phosphorylated eNOS at Ser 1177 (D), and iNOS (E) in gastrocnemius muscle of piglets supplemented with cholesterol and DHA. Tissue lysate proteins (30 µg) were solubilized in
Figure Legend Snippet: Total nNOS (A), phosphorylated nNOS at Ser 852 (B), total eNOS (C), phosphorylated eNOS at Ser 1177 (D), and iNOS (E) in gastrocnemius muscle of piglets supplemented with cholesterol and DHA. Tissue lysate proteins (30 µg) were solubilized in

Techniques Used:

Total nNOS (A), phosphorylated nNOS at Ser 852 (B), total eNOS (C), phosphorylated eNOS at Ser 1177 (D), and iNOS (E) in brain of piglets supplemented with cholesterol and DHA. Tissue lysate proteins (60 µg) were solubilized in SDS-PAGE buffer
Figure Legend Snippet: Total nNOS (A), phosphorylated nNOS at Ser 852 (B), total eNOS (C), phosphorylated eNOS at Ser 1177 (D), and iNOS (E) in brain of piglets supplemented with cholesterol and DHA. Tissue lysate proteins (60 µg) were solubilized in SDS-PAGE buffer

Techniques Used: SDS Page

19) Product Images from "Proinflammatory Role of Angiotensin II in the Aorta of Normotensive Mice"

Article Title: Proinflammatory Role of Angiotensin II in the Aorta of Normotensive Mice

Journal: BioMed Research International

doi: 10.1155/2019/9326896

Immunostaining for inflammatory markers in aorta of mice. Positive staining for IL1- β increased in earlier period (30 minutes) in tunica adventitia and perivascular adipose tissue (PVAT) and in a long period (48 hours) in tunica media, tunica adventitia, and PVAT after angiotensin II injection. Angiotensin II increased immunostaining for TGF- β in acute and late periods in tunica media and later in tunica adventitia. iNOS immunostaining increased in adventitia 48 hours after Ang II injection, while in PVAT increased earlier, after 30 minutes. N=7/group. ∗ ≤0.05; ∗∗ ≤0.01; ∗∗∗ ≤0.001 compared to control.
Figure Legend Snippet: Immunostaining for inflammatory markers in aorta of mice. Positive staining for IL1- β increased in earlier period (30 minutes) in tunica adventitia and perivascular adipose tissue (PVAT) and in a long period (48 hours) in tunica media, tunica adventitia, and PVAT after angiotensin II injection. Angiotensin II increased immunostaining for TGF- β in acute and late periods in tunica media and later in tunica adventitia. iNOS immunostaining increased in adventitia 48 hours after Ang II injection, while in PVAT increased earlier, after 30 minutes. N=7/group. ∗ ≤0.05; ∗∗ ≤0.01; ∗∗∗ ≤0.001 compared to control.

Techniques Used: Immunostaining, Mouse Assay, Staining, Injection

Proinflammatory responses of angiotensin II in aorta of mice. In the presented scheme, Ang II induces a fast macrophage migration to PVAT, possibly increasing iNOS and IL1- β expression. A secondary macrophage migration to tunica media may be related to a late increase of iNOS and IL1- β expression. On the other hand, TGF- β quickly increases in tunica media. The reason why the late expression of TGF- β in adventitia and PVAT could be influenced by earlier TGF- β expression or by macrophages infiltration needs to be investigated.
Figure Legend Snippet: Proinflammatory responses of angiotensin II in aorta of mice. In the presented scheme, Ang II induces a fast macrophage migration to PVAT, possibly increasing iNOS and IL1- β expression. A secondary macrophage migration to tunica media may be related to a late increase of iNOS and IL1- β expression. On the other hand, TGF- β quickly increases in tunica media. The reason why the late expression of TGF- β in adventitia and PVAT could be influenced by earlier TGF- β expression or by macrophages infiltration needs to be investigated.

Techniques Used: Mouse Assay, Migration, Expressing

Photomicrography showing immunostaining for IL1- β in aorta of mice. Positive staining for IL1- β (arrows) increased in earlier period (30 minutes) in tunica adventitia (TA) and perivascular adipose tissue (PVAT) and in a long period (48 hours) in tunica media (TM), tunica adventitia and PVAT after angiotensin II injection (Magnification: 40x).
Figure Legend Snippet: Photomicrography showing immunostaining for IL1- β in aorta of mice. Positive staining for IL1- β (arrows) increased in earlier period (30 minutes) in tunica adventitia (TA) and perivascular adipose tissue (PVAT) and in a long period (48 hours) in tunica media (TM), tunica adventitia and PVAT after angiotensin II injection (Magnification: 40x).

Techniques Used: Immunostaining, Mouse Assay, Staining, Injection

20) Product Images from "Inhibition of poly (ADP-ribose) polymerase and inducible nitric oxide synthase protects against ischemic myocardial damage by reduction of apoptosis"

Article Title: Inhibition of poly (ADP-ribose) polymerase and inducible nitric oxide synthase protects against ischemic myocardial damage by reduction of apoptosis

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2014.2977

The protein expression levels of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). (A) Caspase-3 activity of cardiomyocytes in the various groups. (B) The protein expression levels of cleaved caspase-3 and cleaved PARP, as determined by western blot analysis. (C) Quantitative analysis of cleaved caspase-3 and cleaved PARP, normalized to β-actin. * P
Figure Legend Snippet: The protein expression levels of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). (A) Caspase-3 activity of cardiomyocytes in the various groups. (B) The protein expression levels of cleaved caspase-3 and cleaved PARP, as determined by western blot analysis. (C) Quantitative analysis of cleaved caspase-3 and cleaved PARP, normalized to β-actin. * P

Techniques Used: Expressing, Activity Assay, Western Blot

The protein expression levels of poly (ADP-ribose) (PAR) (active PAR polymerase), and inducible nitric oxide synthase (iNOS), in the myocardium of the various groups. (A) Representative fluorescent images of the myocardium, acquired by laser scanning confocal microscopy (magnification, ×400); PAR staining is green, and 4′, 6-diamidino-2-phenylindole (DAPI) staining for the cell nuclei is blue. (B) Quantitative analysis of PAR, expressed as a fold increase over the control group. (C) The protein expression levels of iNOS were examined by western blot analysis, with the indicated antibody. (D) Quantitative analysis of iNOS, relative to β-actin. Control, n=10; MI, n=7; DPQ, n=8; 1400W, n=8.*P
Figure Legend Snippet: The protein expression levels of poly (ADP-ribose) (PAR) (active PAR polymerase), and inducible nitric oxide synthase (iNOS), in the myocardium of the various groups. (A) Representative fluorescent images of the myocardium, acquired by laser scanning confocal microscopy (magnification, ×400); PAR staining is green, and 4′, 6-diamidino-2-phenylindole (DAPI) staining for the cell nuclei is blue. (B) Quantitative analysis of PAR, expressed as a fold increase over the control group. (C) The protein expression levels of iNOS were examined by western blot analysis, with the indicated antibody. (D) Quantitative analysis of iNOS, relative to β-actin. Control, n=10; MI, n=7; DPQ, n=8; 1400W, n=8.*P

Techniques Used: Expressing, Confocal Microscopy, Staining, Western Blot

21) Product Images from "Alpha-1 Antitrypsin Attenuates M1 Microglia-Mediated Neuroinflammation in Retinal Degeneration"

Article Title: Alpha-1 Antitrypsin Attenuates M1 Microglia-Mediated Neuroinflammation in Retinal Degeneration

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01202

Microglia skewed toward anti-inflammatory M2 phenotype in the presence of alpha-1 antitrypsin (AAT) supplement. (A) In the retinal whole mounts, the amount of CD206 + IBA1 + microglia significantly increased in AAT-treated group compared with the PBS-treated mice, particularly in the central and mid-peripheral retina. (B) Arg1 + cells, another M2 microglia marker, were absent in the PBS-treated rd1 retina, while after AAT supplement, these M2 microglia appeared and most concentrated in the mid-peripheral areas. Scare bar, 50 µm. Depicted is mean ± SEM of three fields/eyes from six eyes. * p
Figure Legend Snippet: Microglia skewed toward anti-inflammatory M2 phenotype in the presence of alpha-1 antitrypsin (AAT) supplement. (A) In the retinal whole mounts, the amount of CD206 + IBA1 + microglia significantly increased in AAT-treated group compared with the PBS-treated mice, particularly in the central and mid-peripheral retina. (B) Arg1 + cells, another M2 microglia marker, were absent in the PBS-treated rd1 retina, while after AAT supplement, these M2 microglia appeared and most concentrated in the mid-peripheral areas. Scare bar, 50 µm. Depicted is mean ± SEM of three fields/eyes from six eyes. * p

Techniques Used: Mouse Assay, Marker

22) Product Images from "Microglial/Macrophage Polarization Dynamics following Traumatic Brain Injury"

Article Title: Microglial/Macrophage Polarization Dynamics following Traumatic Brain Injury

Journal: Journal of Neurotrauma

doi: 10.1089/neu.2015.4268

M1-like and mixed transitional (Mtran) microglial/macrophages predominate the peri-lesional cortex at 7 days post-injury. Representative images and analysis of M1- and M2-like polarized microglia/macrophages after controlled cortical impact (CCI).  (A)  Transforming growth factor (TGF) β+ (red) and CD16/32+ (green) microglia/macrophages in the peri-lesional and distal cortex at 24 h and 7 days post-injury. Insets display M1-like (CD16/32+), M2-like (TGFβ+), and Mtran (CD16/32+/TGFβ+) cells at each time-point. Scale bar = 50 μm.  (B)  Quantification of CD16/32+, TGFβ+, and CD16/32+/TGFβ+ cells at 24 h and 7 days post-injury.  (C)  Arginase 1+ (magenta) and inducible nitric oxide synthase (iNOS)+ (green) microglia/macrophages in the peri-lesional and distal cortex at 24 h and 7 days post-injury. Insets display M1-like (iNOS+) and Mtran (iNOS+/Arg1+) cells at 7 days post-injury. Scale bar = 50 μm  (D)  Quantification of iNOS+, Arg1+, and iNOS+/Arg1+ cells at 24 h and 7 days post-injury. Protein expression levels determined by binary area per region of interest (ROI) (mm 2 ).  n
Figure Legend Snippet: M1-like and mixed transitional (Mtran) microglial/macrophages predominate the peri-lesional cortex at 7 days post-injury. Representative images and analysis of M1- and M2-like polarized microglia/macrophages after controlled cortical impact (CCI). (A) Transforming growth factor (TGF) β+ (red) and CD16/32+ (green) microglia/macrophages in the peri-lesional and distal cortex at 24 h and 7 days post-injury. Insets display M1-like (CD16/32+), M2-like (TGFβ+), and Mtran (CD16/32+/TGFβ+) cells at each time-point. Scale bar = 50 μm. (B) Quantification of CD16/32+, TGFβ+, and CD16/32+/TGFβ+ cells at 24 h and 7 days post-injury. (C) Arginase 1+ (magenta) and inducible nitric oxide synthase (iNOS)+ (green) microglia/macrophages in the peri-lesional and distal cortex at 24 h and 7 days post-injury. Insets display M1-like (iNOS+) and Mtran (iNOS+/Arg1+) cells at 7 days post-injury. Scale bar = 50 μm (D) Quantification of iNOS+, Arg1+, and iNOS+/Arg1+ cells at 24 h and 7 days post-injury. Protein expression levels determined by binary area per region of interest (ROI) (mm 2 ). n

Techniques Used: Expressing

M1-like and M2-like microglial/macrophage activation genes are activated in the ipsilateral cortex after controlled cortical impact (CCI). Real-time polymerase chain reaction (PCR) was used to assess the expression levels of M1- and M2-like microglial/macrophage activation genes in cortex of sham, 1 h, 6 h, 24 h, 72 h, and 7 days CCI mice.  (A)  M1-like genes included interleukin (IL)-1β, IL-12, tumor necrosis factor (TNF) α, inducible nitric oxide synthase (iNOS), and IL-6.  (B)  M2a-like genes included CD206 (mannose receptor), Fizz1, Ym1, (chitinase3-like 3), IL-1rn, and arginase 1.  (C)  M2c-like genes included transforming growth factor (TGF) β, SOCS3 and IL4Rα. Bars represent mean ± standard error of the mean. Statistical analysis by one-way analysis of variance, followed by  post hoc  adjustments using Student Newman Keuls multiple comparison test (* p
Figure Legend Snippet: M1-like and M2-like microglial/macrophage activation genes are activated in the ipsilateral cortex after controlled cortical impact (CCI). Real-time polymerase chain reaction (PCR) was used to assess the expression levels of M1- and M2-like microglial/macrophage activation genes in cortex of sham, 1 h, 6 h, 24 h, 72 h, and 7 days CCI mice. (A) M1-like genes included interleukin (IL)-1β, IL-12, tumor necrosis factor (TNF) α, inducible nitric oxide synthase (iNOS), and IL-6. (B) M2a-like genes included CD206 (mannose receptor), Fizz1, Ym1, (chitinase3-like 3), IL-1rn, and arginase 1. (C) M2c-like genes included transforming growth factor (TGF) β, SOCS3 and IL4Rα. Bars represent mean ± standard error of the mean. Statistical analysis by one-way analysis of variance, followed by post hoc adjustments using Student Newman Keuls multiple comparison test (* p

Techniques Used: Activation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Expressing, Mouse Assay

M2-like polarization of microglia/macrophages after controlled cortical impact (CCI). C57Bl/6 mice were subjected to sham-surgery or CCI, and CD11b+ cells were isolated at 24 h, 72 h, and 7 days post-injury to assess M2-like protein expression by flow cytometry. (A) Representative histograms for M2-like markers transforming growth factor (TGF) β, Ym1, CD206 and arginase 1, include TGFβ high , Ym1 high , CD206 high , and arginase 1 high populations at each time-point. (B) Mean fluorescence intensity (MFI) for TGFβ, % of total TGFβ-positive cells and number of TGFβ high cells. (C) MFI for Ym1, % of total Ym1-positive cells and number of Ym1 high cells. (D) MFI for CD206, % of total CD206-positive cells and number of CD206 high cells. (E) MFI for arginase 1, % of total arginase 1-positive cells and number of arginase 1 high cells. Bars represent mean ± standard error of the mean. Statistical analysis by one-way analysis of variance, followed by post hoc adjustments using Student Newman Keuls multiple comparison test (* p
Figure Legend Snippet: M2-like polarization of microglia/macrophages after controlled cortical impact (CCI). C57Bl/6 mice were subjected to sham-surgery or CCI, and CD11b+ cells were isolated at 24 h, 72 h, and 7 days post-injury to assess M2-like protein expression by flow cytometry. (A) Representative histograms for M2-like markers transforming growth factor (TGF) β, Ym1, CD206 and arginase 1, include TGFβ high , Ym1 high , CD206 high , and arginase 1 high populations at each time-point. (B) Mean fluorescence intensity (MFI) for TGFβ, % of total TGFβ-positive cells and number of TGFβ high cells. (C) MFI for Ym1, % of total Ym1-positive cells and number of Ym1 high cells. (D) MFI for CD206, % of total CD206-positive cells and number of CD206 high cells. (E) MFI for arginase 1, % of total arginase 1-positive cells and number of arginase 1 high cells. Bars represent mean ± standard error of the mean. Statistical analysis by one-way analysis of variance, followed by post hoc adjustments using Student Newman Keuls multiple comparison test (* p

Techniques Used: Mouse Assay, Isolation, Expressing, Flow Cytometry, Cytometry, Fluorescence

NADPH oxidase (NOX2) co-localizes with M1-like and mixed transitional (Mtran) microglia/macrophages at 7 days post-injury. Representative images of NOX2 expression with M1-like, M2-like, and Mtran microglia/macrophages after controlled cortical impact (CCI). (A) gp91 phox + (red), CD16/32+ (magenta), and transforming growth factor (TGF) β+ (green) cells in the peri-lesional cortex (upper panels) and at distant subcortical sites (lower panel). Insets display co-localization of gp91 phox with CD16/32+ and CD16/32+/TGFβ+ cells in peri-lesional regions, and lack of gp91 phox expression in TGFβ+ only cells in distal regions. (B) gp91 phox + (red), iNOS+ (green), and Arg1+ (magenta) cells in the peri-lesional cortex (upper panels) and at distant subcortical sites (lower panel). Insets display co-localization of gp91 phox with inducible nitric oxide synthase (iNOS)+ and iNOS+/Arg1+ cells in peri-lesional regions, and lack of gp91 phox expression in Arg1+ only cells in distal regions. Scale bar = 50 μm. (C) Quantification of co-localization of gp91 phox + with CD16/32+, TGFβ+, and CD16/32+/TGFβ+ in the peri-lesional and distal cortex at 24 h and 7 days post-injury. (D) Quantification of co-localization of gp91 phox + with iNOS+, Arg1+, and iNOS+/Arg1+ in the peri-lesional and distal cortex at 24 h and 7 days post-injury. Protein expression levels determined by binary area per region of interest (ROI; mm 2 ). n
Figure Legend Snippet: NADPH oxidase (NOX2) co-localizes with M1-like and mixed transitional (Mtran) microglia/macrophages at 7 days post-injury. Representative images of NOX2 expression with M1-like, M2-like, and Mtran microglia/macrophages after controlled cortical impact (CCI). (A) gp91 phox + (red), CD16/32+ (magenta), and transforming growth factor (TGF) β+ (green) cells in the peri-lesional cortex (upper panels) and at distant subcortical sites (lower panel). Insets display co-localization of gp91 phox with CD16/32+ and CD16/32+/TGFβ+ cells in peri-lesional regions, and lack of gp91 phox expression in TGFβ+ only cells in distal regions. (B) gp91 phox + (red), iNOS+ (green), and Arg1+ (magenta) cells in the peri-lesional cortex (upper panels) and at distant subcortical sites (lower panel). Insets display co-localization of gp91 phox with inducible nitric oxide synthase (iNOS)+ and iNOS+/Arg1+ cells in peri-lesional regions, and lack of gp91 phox expression in Arg1+ only cells in distal regions. Scale bar = 50 μm. (C) Quantification of co-localization of gp91 phox + with CD16/32+, TGFβ+, and CD16/32+/TGFβ+ in the peri-lesional and distal cortex at 24 h and 7 days post-injury. (D) Quantification of co-localization of gp91 phox + with iNOS+, Arg1+, and iNOS+/Arg1+ in the peri-lesional and distal cortex at 24 h and 7 days post-injury. Protein expression levels determined by binary area per region of interest (ROI; mm 2 ). n

Techniques Used: Expressing

23) Product Images from "MSX3 Switches Microglia Polarization and Protects from Inflammation-Induced Demyelination"

Article Title: MSX3 Switches Microglia Polarization and Protects from Inflammation-Induced Demyelination

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2468-14.2015

MSX3 promotes PPARγ, STAT6, and JAK3 expression in microglia. A–C , MSX3 interacts with promoters of Pparg , Stat6 , and Jak3 in primary cultured microglia. The enrichment of MSX3 on the promoter of the genes indicated was determined using ChIP qPCR in MSX3-overexpressed microglia 72 h after transduction, confirming a direct interaction between MSX3 and promoters of Pparg ( A ), Stat6 ( B ), and Jak3 ( C ). In contrast, no direct interaction between MSX3 and promoters of Jak1 , Ptp1b , or Cebpb was detected. The data are shown as the means ± SEM of three independent experiments. ** p
Figure Legend Snippet: MSX3 promotes PPARγ, STAT6, and JAK3 expression in microglia. A–C , MSX3 interacts with promoters of Pparg , Stat6 , and Jak3 in primary cultured microglia. The enrichment of MSX3 on the promoter of the genes indicated was determined using ChIP qPCR in MSX3-overexpressed microglia 72 h after transduction, confirming a direct interaction between MSX3 and promoters of Pparg ( A ), Stat6 ( B ), and Jak3 ( C ). In contrast, no direct interaction between MSX3 and promoters of Jak1 , Ptp1b , or Cebpb was detected. The data are shown as the means ± SEM of three independent experiments. ** p

Techniques Used: Expressing, Cell Culture, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Transduction

24) Product Images from "Cell-Specific Nitric Oxide Synthase-Isoenzyme Expression and Regulation in Response to Endotoxin in Intact Rat Lungs"

Article Title: Cell-Specific Nitric Oxide Synthase-Isoenzyme Expression and Regulation in Response to Endotoxin in Intact Rat Lungs

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

doi: 10.1038/labinvest.3780436

Immunohistochemical localization of neuronal/brain nitric oxide synthase (bNOS) (A), inducible (i)NOS (B) and endothelial (e)NOS (C) in normal rat lung tissue. (A). Strong bNOS immunostaining was detected in bronchial epithelial cells of small bronchioli within the alveolar tissue. (B) Constitutive iNOS expression was found in bronchial epithelium and bronchial smooth muscle cells. Vascular smooth muscle cells (VSMCs) and especially endothelial cells of pulmonary vessels showed positive immunostaining for eNOS (C). Control staining with nonspecific immune serum shows no staining reaction (D).
Figure Legend Snippet: Immunohistochemical localization of neuronal/brain nitric oxide synthase (bNOS) (A), inducible (i)NOS (B) and endothelial (e)NOS (C) in normal rat lung tissue. (A). Strong bNOS immunostaining was detected in bronchial epithelial cells of small bronchioli within the alveolar tissue. (B) Constitutive iNOS expression was found in bronchial epithelium and bronchial smooth muscle cells. Vascular smooth muscle cells (VSMCs) and especially endothelial cells of pulmonary vessels showed positive immunostaining for eNOS (C). Control staining with nonspecific immune serum shows no staining reaction (D).

Techniques Used: Immunohistochemistry, Immunostaining, Expressing, Staining

25) Product Images from "Indole-3-Carbinol Derivative DIM Mitigates Carbon Tetrachloride-Induced Acute Liver Injury in Mice by Inhibiting Inflammatory Response, Apoptosis and Regulating Oxidative Stress"

Article Title: Indole-3-Carbinol Derivative DIM Mitigates Carbon Tetrachloride-Induced Acute Liver Injury in Mice by Inhibiting Inflammatory Response, Apoptosis and Regulating Oxidative Stress

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21062048

DIM pretreatment inhibits the protein expression levels of inflammatory factors in liver tissue of CCl 4 -induced liver injury in mice. ( A ) Protein expression of pro-inflammatory cytokines (TNF-α, IL-6 IL-1β) and inflammatory mediators (COX-2 and iNOS) at 24 h after CCl 4 injection by using Western blot analysis. ( B ) Quantification of relative protein expression normalized to β-actin. ( C ) The level of serum TNF-α measured using ELISA kits. The values represent the mean ± SD ( n = 3). ### p
Figure Legend Snippet: DIM pretreatment inhibits the protein expression levels of inflammatory factors in liver tissue of CCl 4 -induced liver injury in mice. ( A ) Protein expression of pro-inflammatory cytokines (TNF-α, IL-6 IL-1β) and inflammatory mediators (COX-2 and iNOS) at 24 h after CCl 4 injection by using Western blot analysis. ( B ) Quantification of relative protein expression normalized to β-actin. ( C ) The level of serum TNF-α measured using ELISA kits. The values represent the mean ± SD ( n = 3). ### p

Techniques Used: Expressing, Mouse Assay, Injection, Western Blot, Enzyme-linked Immunosorbent Assay

26) Product Images from "Dual Angiotensin Receptor and Neprilysin Inhibitor Ameliorates Portal Hypertension in Portal Hypertensive Rats"

Article Title: Dual Angiotensin Receptor and Neprilysin Inhibitor Ameliorates Portal Hypertension in Portal Hypertensive Rats

Journal: Pharmaceutics

doi: 10.3390/pharmaceutics12040320

Hepatic protein expressions of control, valsartan-, LCZ696-treated PVL rats. The densitometric quantification and representative Western blot images are shown. The upper panel reveals that the endothelial-1 (ET-1) protein expression was significantly downregulated by LCZ696 compared to the control group. The vascular endothelial growth factor (VEGF), cyclooxygenase (COX)-1, and COX-2 protein expressions were not significantly different among control, valsartan-, and LCZ696-treated PVL rats. The lower panel indicates that the phosphorylated-endothelial nitric oxide synthase (eNOS) protein expressions were downregulated by valsartan and LCZ696 treatments. The phosphorylated-nuclear factor kappa B (NFκB) p65, phosphorylated-antinuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and phosphorylated-inducible nitric oxide synthase (iNOS) protein expressions were not affected by valsartan and LCZ696.
Figure Legend Snippet: Hepatic protein expressions of control, valsartan-, LCZ696-treated PVL rats. The densitometric quantification and representative Western blot images are shown. The upper panel reveals that the endothelial-1 (ET-1) protein expression was significantly downregulated by LCZ696 compared to the control group. The vascular endothelial growth factor (VEGF), cyclooxygenase (COX)-1, and COX-2 protein expressions were not significantly different among control, valsartan-, and LCZ696-treated PVL rats. The lower panel indicates that the phosphorylated-endothelial nitric oxide synthase (eNOS) protein expressions were downregulated by valsartan and LCZ696 treatments. The phosphorylated-nuclear factor kappa B (NFκB) p65, phosphorylated-antinuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and phosphorylated-inducible nitric oxide synthase (iNOS) protein expressions were not affected by valsartan and LCZ696.

Techniques Used: Western Blot, Expressing

27) Product Images from "Anthocyanins abrogate glutamate-induced AMPK activation, oxidative stress, neuroinflammation, and neurodegeneration in postnatal rat brain"

Article Title: Anthocyanins abrogate glutamate-induced AMPK activation, oxidative stress, neuroinflammation, and neurodegeneration in postnatal rat brain

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-016-0752-y

Glutamate-induced toxicity in SH-SY5Y and BV2 cells is AMPK dependent. a The Western blot analysis and relative integrated density histograms of p-NF- k B, COX2 and caspase-3 proteins in SH-SY5Y cells. The treatment details are the same as described in the “ Methods ” section. β-Actin was used as a loading control. b The activity histogram of NF- k Bp65 (total) in the cell lysates, as measured with an ELISA kit. c The COX2 assay was performed with SH-SY5Y cells after treatment with the indicated concentrations of glutamate, anthocyanins, and compound C for the indicated duration of time. After treatment, cells were fixed prior to performing the assay as per the manufacturer’s instructions. The immunostaining images ( d ) of p-NF- k B ( green ), p-AMPK ( red ) ( e ), Nrf2 ( green ) and DAPI ( blue ) in the BV2 cells treated with glutamate and anthocyanins. Their densities are depicted in the relative IOD histogram. f The COX2 assay histogram in BV2 cells was conducted according to the manufacturer’s instructions. Significance, *** P
Figure Legend Snippet: Glutamate-induced toxicity in SH-SY5Y and BV2 cells is AMPK dependent. a The Western blot analysis and relative integrated density histograms of p-NF- k B, COX2 and caspase-3 proteins in SH-SY5Y cells. The treatment details are the same as described in the “ Methods ” section. β-Actin was used as a loading control. b The activity histogram of NF- k Bp65 (total) in the cell lysates, as measured with an ELISA kit. c The COX2 assay was performed with SH-SY5Y cells after treatment with the indicated concentrations of glutamate, anthocyanins, and compound C for the indicated duration of time. After treatment, cells were fixed prior to performing the assay as per the manufacturer’s instructions. The immunostaining images ( d ) of p-NF- k B ( green ), p-AMPK ( red ) ( e ), Nrf2 ( green ) and DAPI ( blue ) in the BV2 cells treated with glutamate and anthocyanins. Their densities are depicted in the relative IOD histogram. f The COX2 assay histogram in BV2 cells was conducted according to the manufacturer’s instructions. Significance, *** P

Techniques Used: Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunostaining

Glutamate-induced neurotoxicity in the developing rodent brain is AMPK dependent. a The expression level of p-AMPK, Nrf2, and HO-1 proteins in the P7 rat brain homogenates that were treated with glutamate or glutamate with or without compound C and anthocyanins for 4 h. Their densities were measured with the help of Sigma Gel software. Their respective relative density histograms were made with Prism GraphPad. The membranes were redeveloped for β-actin and used as a loading control. b , c The histograms of AMPK activity and ROS assay were conducted with brain homogenates of the abovementioned experimental animal groups. The assays were performed in triplicate with the same results. d The Western blot analysis of p-NF- k B, COX2, and caspase-3 proteins in the hippocampus of postnatal day 7 rat brain following treatment with glutamate, glutamate and compound C, or glutamate, compound C, and anthocyanins. The relative integrated density for the abovementioned proteins are depicted in the histograms. The density values are expressed in arbitrary units as the mean ± SEM for the indicated proteins ( n = 5 animals per group). Details are provided in the “ Methods ” section. e The activity histogram of NF- k Bp65 (total) in the brain homogenates of treated animal’s measured with ELISA kit method. Significance; *** P
Figure Legend Snippet: Glutamate-induced neurotoxicity in the developing rodent brain is AMPK dependent. a The expression level of p-AMPK, Nrf2, and HO-1 proteins in the P7 rat brain homogenates that were treated with glutamate or glutamate with or without compound C and anthocyanins for 4 h. Their densities were measured with the help of Sigma Gel software. Their respective relative density histograms were made with Prism GraphPad. The membranes were redeveloped for β-actin and used as a loading control. b , c The histograms of AMPK activity and ROS assay were conducted with brain homogenates of the abovementioned experimental animal groups. The assays were performed in triplicate with the same results. d The Western blot analysis of p-NF- k B, COX2, and caspase-3 proteins in the hippocampus of postnatal day 7 rat brain following treatment with glutamate, glutamate and compound C, or glutamate, compound C, and anthocyanins. The relative integrated density for the abovementioned proteins are depicted in the histograms. The density values are expressed in arbitrary units as the mean ± SEM for the indicated proteins ( n = 5 animals per group). Details are provided in the “ Methods ” section. e The activity histogram of NF- k Bp65 (total) in the brain homogenates of treated animal’s measured with ELISA kit method. Significance; *** P

Techniques Used: Expressing, Software, Activity Assay, ROS Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Anthocyanins reduced glutamate-induced glial cell activation, neuroinflammation, and DNA damage in hippocampal CA1 region of the developing rat brain. Given are representative immunofluorescence images along with relative IOD histograms of a astrocytes (GFAP)-positive cells and b microglia (Iba-1)-positive cells in the CA1 region of experimental groups. Images represent immunostaining performed with tissue sections prepared from at least five rats in each group. Panels representing hippocampal CA1 region immunostained with GFAP ( green ), Iba-1( red ) counterstained with DAPI (blue) of young rat brain. c The immunoblot represents proinflammatory markers, including p-NF- k B, COX2 and TNF-α proteins, which are accompanied by their relative density histograms. d The ELISA histogram of NF- k Bp65 (total) in the brain homogenates of treated animals. The assay was repeated three times according to the manufacturer’s instruction. e The extent of DNA damage in the CA1 region by glutamate was analyzed by conducting TUNEL assay. To quantify DNA damage, ImageJ and Prism GraphPad programs were used. The values represent the mean ± SEM for the indicated proteins ( n = 5 animals per group). Significance, *** P
Figure Legend Snippet: Anthocyanins reduced glutamate-induced glial cell activation, neuroinflammation, and DNA damage in hippocampal CA1 region of the developing rat brain. Given are representative immunofluorescence images along with relative IOD histograms of a astrocytes (GFAP)-positive cells and b microglia (Iba-1)-positive cells in the CA1 region of experimental groups. Images represent immunostaining performed with tissue sections prepared from at least five rats in each group. Panels representing hippocampal CA1 region immunostained with GFAP ( green ), Iba-1( red ) counterstained with DAPI (blue) of young rat brain. c The immunoblot represents proinflammatory markers, including p-NF- k B, COX2 and TNF-α proteins, which are accompanied by their relative density histograms. d The ELISA histogram of NF- k Bp65 (total) in the brain homogenates of treated animals. The assay was repeated three times according to the manufacturer’s instruction. e The extent of DNA damage in the CA1 region by glutamate was analyzed by conducting TUNEL assay. To quantify DNA damage, ImageJ and Prism GraphPad programs were used. The values represent the mean ± SEM for the indicated proteins ( n = 5 animals per group). Significance, *** P

Techniques Used: Activation Assay, Immunofluorescence, Immunostaining, Enzyme-linked Immunosorbent Assay, TUNEL Assay

28) Product Images from "Molecular Study of Dietary Heptadecane for the Anti-Inflammatory Modulation of NF-kB in the Aged Kidney"

Article Title: Molecular Study of Dietary Heptadecane for the Anti-Inflammatory Modulation of NF-kB in the Aged Kidney

Journal: PLoS ONE

doi: 10.1371/journal.pone.0059316

Possible mechanism of the effect of aging and heptadecane on the NIK/MAPKs/NF-kB pathway. RS, reactive species; NF-kB, nuclear factor kappa B; NIK, NF-kB-inducing kinase; IKK, IkB kinase; COX-2, cyclooxygenase-2.R.
Figure Legend Snippet: Possible mechanism of the effect of aging and heptadecane on the NIK/MAPKs/NF-kB pathway. RS, reactive species; NF-kB, nuclear factor kappa B; NIK, NF-kB-inducing kinase; IKK, IkB kinase; COX-2, cyclooxygenase-2.R.

Techniques Used:

29) Product Images from "Ischemic postconditioning protects the heart against ischemia–reperfusion injury via neuronal nitric oxide synthase in the sarcoplasmic reticulum and mitochondria"

Article Title: Ischemic postconditioning protects the heart against ischemia–reperfusion injury via neuronal nitric oxide synthase in the sarcoplasmic reticulum and mitochondria

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.108

Expression of arginase II, p-AMPK Thr172 and β -actin in the myocardium at 30 min of reperfusion. ( a ) Arginase II expression was significantly increased in the I/R group; this increase was downregulated by IPostC. ( b and c ) p-AMPK Thr172 expression was increased in the IPostC group; this increase was abolished by nNOS inhibitors and nNOS siRNA ( n =3 per group). P
Figure Legend Snippet: Expression of arginase II, p-AMPK Thr172 and β -actin in the myocardium at 30 min of reperfusion. ( a ) Arginase II expression was significantly increased in the I/R group; this increase was downregulated by IPostC. ( b and c ) p-AMPK Thr172 expression was increased in the IPostC group; this increase was abolished by nNOS inhibitors and nNOS siRNA ( n =3 per group). P

Techniques Used: Expressing

PGC-1 α expression and SOD activity in the myocardium and H9C2 cells in vitro . IPostC increased PGC-1 α mRNA levels ( c and d ) and protein expression ( a and b ); these effects were abolished by the AMPK inhibitor compound C. ( e ) IPostC recovered SOD activity, which was abolished by AMPK inhibition ( n =4 per group). P
Figure Legend Snippet: PGC-1 α expression and SOD activity in the myocardium and H9C2 cells in vitro . IPostC increased PGC-1 α mRNA levels ( c and d ) and protein expression ( a and b ); these effects were abolished by the AMPK inhibitor compound C. ( e ) IPostC recovered SOD activity, which was abolished by AMPK inhibition ( n =4 per group). P

Techniques Used: Pyrolysis Gas Chromatography, Expressing, Activity Assay, In Vitro, Inhibition

IPostC significantly protects hearts against I/R injury, but its molecular mechanisms remain poorly understood. The hypothesis: IPostC regulates uncoupled nNOS and the nNOS/AMPK/PGC-1 α axis to decrease oxidative stress and improves SR function by increasing PLB phosphorylation via a nNOS-mediated pathway to decreased intracellular Ca 2+ overload, which protects hearts against I/R injury. Considering that the effects of nNOS are closely associated with myocardial I/R injury, nNOS may thus be a promising future therapeutic target for ischemic heart disease
Figure Legend Snippet: IPostC significantly protects hearts against I/R injury, but its molecular mechanisms remain poorly understood. The hypothesis: IPostC regulates uncoupled nNOS and the nNOS/AMPK/PGC-1 α axis to decrease oxidative stress and improves SR function by increasing PLB phosphorylation via a nNOS-mediated pathway to decreased intracellular Ca 2+ overload, which protects hearts against I/R injury. Considering that the effects of nNOS are closely associated with myocardial I/R injury, nNOS may thus be a promising future therapeutic target for ischemic heart disease

Techniques Used: Pyrolysis Gas Chromatography

30) Product Images from "SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI)"

Article Title: SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI)

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-9-237

S - nitrosylation of PDI in astrocytes following OGD / reperfusion. A . In the presence of both ascorbate and biotin-HPDP, astrocytes following OGD 8 h/reperfusion 24 h treatment had detectable SNO-PDI, indicating the specificity of biotin-switch assay. B . PDI was S-nitrosylated in cultured astrocytes following OGD/reperfusion. Densitometric quantitation showed significant differences of SNO-PDI levels between the control group and the OGD/reperfusion groups. C . 1400W significantly inhibited NO production; the iNOS protein expression remained unaltered in cultured astrocytes following OGD 8 h/reperfusion 24 h. The cultured astrocytes were pretreated with various concentrations of iNOS inhibitor 1400W, which suppressed the S-nitrosylated PDI formation under OGD 8 h/reperfusion 24 h. The level of SNO-PDI was reduced with the increased concentration of 1400W (1, 10, and 50 μM). Moreover, 1400W suppressed S-nitrosylation of PDI, with the maximum effect seen at the concentration of 50 μM. Three independent experiments were performed. Data were presented as mean ± SEM; *, P
Figure Legend Snippet: S - nitrosylation of PDI in astrocytes following OGD / reperfusion. A . In the presence of both ascorbate and biotin-HPDP, astrocytes following OGD 8 h/reperfusion 24 h treatment had detectable SNO-PDI, indicating the specificity of biotin-switch assay. B . PDI was S-nitrosylated in cultured astrocytes following OGD/reperfusion. Densitometric quantitation showed significant differences of SNO-PDI levels between the control group and the OGD/reperfusion groups. C . 1400W significantly inhibited NO production; the iNOS protein expression remained unaltered in cultured astrocytes following OGD 8 h/reperfusion 24 h. The cultured astrocytes were pretreated with various concentrations of iNOS inhibitor 1400W, which suppressed the S-nitrosylated PDI formation under OGD 8 h/reperfusion 24 h. The level of SNO-PDI was reduced with the increased concentration of 1400W (1, 10, and 50 μM). Moreover, 1400W suppressed S-nitrosylation of PDI, with the maximum effect seen at the concentration of 50 μM. Three independent experiments were performed. Data were presented as mean ± SEM; *, P

Techniques Used: Biotin Switch Assay, Cell Culture, Quantitation Assay, Expressing, Concentration Assay

31) Product Images from "Interferon regulatory factor 3 inhibits astrocyte inflammatory gene expression through suppression of the proinflammatory miR-155 and miR-155 *"

Article Title: Interferon regulatory factor 3 inhibits astrocyte inflammatory gene expression through suppression of the proinflammatory miR-155 and miR-155 *

Journal: Glia

doi: 10.1002/glia.21233

Astrocyte iNOS expression is inhibited by Ad-IRF3
Figure Legend Snippet: Astrocyte iNOS expression is inhibited by Ad-IRF3

Techniques Used: Expressing

32) Product Images from "Glucocorticoids induce production of reactive oxygen species/reactive nitrogen species and DNA damage through an iNOS mediated pathway in breast cancer"

Article Title: Glucocorticoids induce production of reactive oxygen species/reactive nitrogen species and DNA damage through an iNOS mediated pathway in breast cancer

Journal: Breast Cancer Research : BCR

doi: 10.1186/s13058-017-0823-8

Expression of inducible nitric oxide synthase (iNOS) is increased in breast carcinoma and mouse mammary tumours in response to stress. a Oncomine Cancer Mircoarray databases were used to analyse expression of NOS2 in the The Cancer Genome Atlas ( TGCA ) Breast database ( n = 137). Expression was compared between normal breast tissue ( n = 61) and invasive breast carcinoma ( n = 79). b MCF-7 cells were exposed to cortisol (1 μM) for 30 minutes and 24 h and mRNA was extracted. cDNA was synthesised and amplified in the presence of gene-specific primers for NOS2 and β-actin using qRT-PCR. Cycle threshold (Ct) values for NOS2 were normalised against β-actin and fold change was calculated using the delta-Ct method. Data are mean ± SEM and significance was determined using one-way analysis of variance (post hoc Tukey multiple comparisons). c The 4T1 mouse mammary gland cells were transplanted into the fourth mammary fat pad of female BALB/C mice and the animals were randomised into groups exposed to either acute restrain stress or no stress. Tumours were harvested, fixed in paraffin and sectioned subsequent to immunohistochemical detection ( IHC ) of iNOS. Labelling was scored (0–3) according to intensity; representative panels are shown. The Mann-Whitney test was used to ascertain statistical significance. *Significant increase: * p
Figure Legend Snippet: Expression of inducible nitric oxide synthase (iNOS) is increased in breast carcinoma and mouse mammary tumours in response to stress. a Oncomine Cancer Mircoarray databases were used to analyse expression of NOS2 in the The Cancer Genome Atlas ( TGCA ) Breast database ( n = 137). Expression was compared between normal breast tissue ( n = 61) and invasive breast carcinoma ( n = 79). b MCF-7 cells were exposed to cortisol (1 μM) for 30 minutes and 24 h and mRNA was extracted. cDNA was synthesised and amplified in the presence of gene-specific primers for NOS2 and β-actin using qRT-PCR. Cycle threshold (Ct) values for NOS2 were normalised against β-actin and fold change was calculated using the delta-Ct method. Data are mean ± SEM and significance was determined using one-way analysis of variance (post hoc Tukey multiple comparisons). c The 4T1 mouse mammary gland cells were transplanted into the fourth mammary fat pad of female BALB/C mice and the animals were randomised into groups exposed to either acute restrain stress or no stress. Tumours were harvested, fixed in paraffin and sectioned subsequent to immunohistochemical detection ( IHC ) of iNOS. Labelling was scored (0–3) according to intensity; representative panels are shown. The Mann-Whitney test was used to ascertain statistical significance. *Significant increase: * p

Techniques Used: Expressing, Amplification, Quantitative RT-PCR, Mouse Assay, Immunohistochemistry, MANN-WHITNEY

33) Product Images from "Activation of M1 macrophages plays a critical role in the initiation of acute lung injury"

Article Title: Activation of M1 macrophages plays a critical role in the initiation of acute lung injury

Journal: Bioscience Reports

doi: 10.1042/BSR20171555

M1 and M2 macrophages are significantly induced after LPS treatment ( A ) Real-time PCR analysis showed that the RNA levels of IL-1β and iNOS (markers of M1 macrophages), and IL-10 and CD206 (markers of M2 macrophages) were significantly increased in a time-dependent manner after LPS treatment. ( B ) Western blot analysis showed that the protein levels of IL-1β, iNOS, IL-10, and CD206 were significantly increased in a time-dependent manner. ( C ) The CD68+ inflammatory cells were significantly increased in the lungs after LPS treatment. ( D ) The M1 macrophages (iNOS+) were significantly increased within 3–24 h after LPS treatment. ( E ) The M2 macrophages (CD206+) were significantly increased within 10 h to 24 h after LPS treatment; * P
Figure Legend Snippet: M1 and M2 macrophages are significantly induced after LPS treatment ( A ) Real-time PCR analysis showed that the RNA levels of IL-1β and iNOS (markers of M1 macrophages), and IL-10 and CD206 (markers of M2 macrophages) were significantly increased in a time-dependent manner after LPS treatment. ( B ) Western blot analysis showed that the protein levels of IL-1β, iNOS, IL-10, and CD206 were significantly increased in a time-dependent manner. ( C ) The CD68+ inflammatory cells were significantly increased in the lungs after LPS treatment. ( D ) The M1 macrophages (iNOS+) were significantly increased within 3–24 h after LPS treatment. ( E ) The M2 macrophages (CD206+) were significantly increased within 10 h to 24 h after LPS treatment; * P

Techniques Used: Real-time Polymerase Chain Reaction, Western Blot

34) Product Images from "Specific endothelial heparin-binding EGF-like growth factor deletion ameliorates renal injury induced by chronic angiotensin II infusion"

Article Title: Specific endothelial heparin-binding EGF-like growth factor deletion ameliorates renal injury induced by chronic angiotensin II infusion

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.00377.2015

Glomerular injury and apoptosis in the kidneys of saline- or Ang II-infused mice. A , top : glomerular capillary density shown by Von Willebrand factor (vWF) immunostaining; middle : podocytes shown by WT1 staining; bottom : apoptosis shown by cleaved caspase-3
Figure Legend Snippet: Glomerular injury and apoptosis in the kidneys of saline- or Ang II-infused mice. A , top : glomerular capillary density shown by Von Willebrand factor (vWF) immunostaining; middle : podocytes shown by WT1 staining; bottom : apoptosis shown by cleaved caspase-3

Techniques Used: Mouse Assay, Immunostaining, Staining

35) Product Images from "JUNB/AP-1 controls IFN-? during inflammatory liver disease"

Article Title: JUNB/AP-1 controls IFN-? during inflammatory liver disease

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI70405

STAT1 targets, IRF1 and iNos, are altered in Junb Δ li* mice.
Figure Legend Snippet: STAT1 targets, IRF1 and iNos, are altered in Junb Δ li* mice.

Techniques Used: Mouse Assay

Restoring the IFN-γ/pSTAT1/IRF1 pathway increases liver damage.
Figure Legend Snippet: Restoring the IFN-γ/pSTAT1/IRF1 pathway increases liver damage.

Techniques Used:

36) Product Images from "Role of Host Protein Tyrosine Phosphatase SHP-1 in Leishmania donovani-Induced Inhibition of Nitric Oxide Production "

Article Title: Role of Host Protein Tyrosine Phosphatase SHP-1 in Leishmania donovani-Induced Inhibition of Nitric Oxide Production

Journal:

doi: 10.1128/IAI.00853-05

STAT1 nuclear translocation upon IFN-γ stimulation in infected and uninfected SHP-1-deficient macrophages and littermate control cells. Uninfected (Nil) or L. donovani -infected (Ld) SHP-1-deficient or littermate macrophages were stimulated with
Figure Legend Snippet: STAT1 nuclear translocation upon IFN-γ stimulation in infected and uninfected SHP-1-deficient macrophages and littermate control cells. Uninfected (Nil) or L. donovani -infected (Ld) SHP-1-deficient or littermate macrophages were stimulated with

Techniques Used: Translocation Assay, Infection

37) Product Images from "Polarization of macrophages in the tumor microenvironment is influenced by EGFR signaling within colon cancer cells"

Article Title: Polarization of macrophages in the tumor microenvironment is influenced by EGFR signaling within colon cancer cells

Journal: Oncotarget

doi: 10.18632/oncotarget.12207

Cetuximab modulates macrophage polarization in an AOM/DSS mouse model A. Establishment of the AOM/DSS mouse model. AOM was injected intraperitoneally at 12.5 mg/kg body weight. After one week, mice were given drinking water containing 2.5% DSS for 5 days followed by 16 days of regular drinking water. After two cycles of DSS treatment, cetuximab (1 mg/mouse, twice a week) was injected intraperitoneally for a month, and the mice were then sacrificed. B. Representative images of colon tumors in normal (right), AOM/DSS (2AD) (middle), and cetuximab-treated AOM/DSS mice (2AD+cetu) (left). C. Tumor quantification. Cetuximab treatment (2AD + cetu) reduced tumor numbers compared to 2AD mice. D. p-EGFR (Y1068), EGFR, Arg1, and iNOS protein levels in normal mice, 2AD and 2AD+cetu mouse tumors were detected by Western blot. All experiments were repeated three times. E. Representative photomicrographs of immunostaining for p-EGFR (Y1068), PCNA, F4/80, Arg1, and iNOS. Scale bars: 25 μm. F. M1 marker (IL-12, iNOS) and M2 marker (IL-4, IL-10, Arg1) mRNA levels in normal mouse colon tissues, 2AD and 2AD+cetu mouse tumor tissues were evaluated by q-PCR. G. Percentages of CD11b + /F4/80 + and F4/80 + /CD206 + cells in normal, 2AD, and 2AD+cetu mice colon tissues were detected by flow cytometry. Colon tissues were cut into small pieces (1-2 mm) and incubated with collagenase D (1- mg/mL), dispase II (1 mg/mL), and DNase I (100 μg/mL) for 30-45 min in a shaking incubator at 37°C, and single-cell suspensions were then incubated with antibodies. Bars represent means ± SD (n = 3) for each treatment. * p
Figure Legend Snippet: Cetuximab modulates macrophage polarization in an AOM/DSS mouse model A. Establishment of the AOM/DSS mouse model. AOM was injected intraperitoneally at 12.5 mg/kg body weight. After one week, mice were given drinking water containing 2.5% DSS for 5 days followed by 16 days of regular drinking water. After two cycles of DSS treatment, cetuximab (1 mg/mouse, twice a week) was injected intraperitoneally for a month, and the mice were then sacrificed. B. Representative images of colon tumors in normal (right), AOM/DSS (2AD) (middle), and cetuximab-treated AOM/DSS mice (2AD+cetu) (left). C. Tumor quantification. Cetuximab treatment (2AD + cetu) reduced tumor numbers compared to 2AD mice. D. p-EGFR (Y1068), EGFR, Arg1, and iNOS protein levels in normal mice, 2AD and 2AD+cetu mouse tumors were detected by Western blot. All experiments were repeated three times. E. Representative photomicrographs of immunostaining for p-EGFR (Y1068), PCNA, F4/80, Arg1, and iNOS. Scale bars: 25 μm. F. M1 marker (IL-12, iNOS) and M2 marker (IL-4, IL-10, Arg1) mRNA levels in normal mouse colon tissues, 2AD and 2AD+cetu mouse tumor tissues were evaluated by q-PCR. G. Percentages of CD11b + /F4/80 + and F4/80 + /CD206 + cells in normal, 2AD, and 2AD+cetu mice colon tissues were detected by flow cytometry. Colon tissues were cut into small pieces (1-2 mm) and incubated with collagenase D (1- mg/mL), dispase II (1 mg/mL), and DNase I (100 μg/mL) for 30-45 min in a shaking incubator at 37°C, and single-cell suspensions were then incubated with antibodies. Bars represent means ± SD (n = 3) for each treatment. * p

Techniques Used: Injection, Mouse Assay, Western Blot, Immunostaining, Marker, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Incubation

38) Product Images from "Andrographolide Enhances Nuclear Factor-?B Subunit p65 Ser536 Dephosphorylation through Activation of Protein Phosphatase 2A in Vascular Smooth Muscle Cells *"

Article Title: Andrographolide Enhances Nuclear Factor-?B Subunit p65 Ser536 Dephosphorylation through Activation of Protein Phosphatase 2A in Vascular Smooth Muscle Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.123968

Effects of andrographolide on NF-κB activation in LPS/IFN-γ-stimulated rat VSMCs. A , cells were pretreated with vehicle or andrographolide for 30 min before treatment with the combination of LPS (50 μg/ml) and IFN-γ (100 units/ml) for another 30 min. Following incubation, the nuclear protein fraction was prepared for the oligonucleotide pull-down assay. The input extracts were normalized between samples by analyzing the levels of HDAC3. Data are presented as means ± S.E. ( error bars ). *, p
Figure Legend Snippet: Effects of andrographolide on NF-κB activation in LPS/IFN-γ-stimulated rat VSMCs. A , cells were pretreated with vehicle or andrographolide for 30 min before treatment with the combination of LPS (50 μg/ml) and IFN-γ (100 units/ml) for another 30 min. Following incubation, the nuclear protein fraction was prepared for the oligonucleotide pull-down assay. The input extracts were normalized between samples by analyzing the levels of HDAC3. Data are presented as means ± S.E. ( error bars ). *, p

Techniques Used: Activation Assay, Incubation, Pull Down Assay

39) Product Images from "Endothelin-1 in osteoarthritic chondrocytes triggers nitric oxide production and upregulates collagenase production"

Article Title: Endothelin-1 in osteoarthritic chondrocytes triggers nitric oxide production and upregulates collagenase production

Journal: Arthritis Research & Therapy

doi: 10.1186/ar1489

Phosphorylation of p38 mitogen-activated protein (MAP) kinase, Akt, p44/42 and stress-activated protein kinase/Jun-N-terminal kinase (SAP/JNK) by endothelin-1 (ET-1) in human osteoarthritis (OA) chondrocytes. (a) Western immunoblot of p38 MAP kinase. Confluent human OA chondrocytes were incubated with ET-1 (10 nM) for 10 or 45 min and the cell extracts were prepared as described in Materials and methods. Western immunoblots used antiserum against activated (phospho-p38) and total p38 MAP kinase (p38 T). Representative result of three different experiments. (b) Western immunoblot of Akt. Cells were incubated for 2, 5, 15, 30, 45 or 60 min in the presence of ET-1 (10 nM) and cell extracts were prepared as described in Materials and methods. Western immunoblot was carried out using an antiserum specific for phospho Ser 473 of Akt. Representative result of three different experiments. (c) Western immunoblot of p44/42. Confluent human OA chondrocytes were incubated with ET-1 (10 nM) for 0, 5, 15 or 60 min and cell extracts were prepared as described in Materials and methods. (d) Western immunoblot of SAP/JNK protein kinase. Confluent human OA chondrocytes were incubated with ET-1 (10 nM) for 0, 5, 30, 45 and 60 min, and cell extracts were prepared as described in Materials and methods. Actin detection was used as a control of the level of proteins loaded. Representative blot of three independent experiments.
Figure Legend Snippet: Phosphorylation of p38 mitogen-activated protein (MAP) kinase, Akt, p44/42 and stress-activated protein kinase/Jun-N-terminal kinase (SAP/JNK) by endothelin-1 (ET-1) in human osteoarthritis (OA) chondrocytes. (a) Western immunoblot of p38 MAP kinase. Confluent human OA chondrocytes were incubated with ET-1 (10 nM) for 10 or 45 min and the cell extracts were prepared as described in Materials and methods. Western immunoblots used antiserum against activated (phospho-p38) and total p38 MAP kinase (p38 T). Representative result of three different experiments. (b) Western immunoblot of Akt. Cells were incubated for 2, 5, 15, 30, 45 or 60 min in the presence of ET-1 (10 nM) and cell extracts were prepared as described in Materials and methods. Western immunoblot was carried out using an antiserum specific for phospho Ser 473 of Akt. Representative result of three different experiments. (c) Western immunoblot of p44/42. Confluent human OA chondrocytes were incubated with ET-1 (10 nM) for 0, 5, 15 or 60 min and cell extracts were prepared as described in Materials and methods. (d) Western immunoblot of SAP/JNK protein kinase. Confluent human OA chondrocytes were incubated with ET-1 (10 nM) for 0, 5, 30, 45 and 60 min, and cell extracts were prepared as described in Materials and methods. Actin detection was used as a control of the level of proteins loaded. Representative blot of three independent experiments.

Techniques Used: Western Blot, Incubation

40) Product Images from "Scolopendra subspinipes mutilans Extract Suppresses Inflammatory and Neuropathic Pain In Vitro and In Vivo"

Article Title: Scolopendra subspinipes mutilans Extract Suppresses Inflammatory and Neuropathic Pain In Vitro and In Vivo

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2018/5057372

Effects of SSM on SNCI-induced protein expression of TNF-α, IL-6, iNOS, and COX-2 . The sciatic nerve was crushed for 30 s using a surgical clip. (a) Relative protein expression levels of TNF- α and IL-6. (b) Relative protein expression levels of iNOS and COX-2. Bands were detected using an enhanced chemiluminescence (ECL) detection kit. Actin was used as an internal control (46 kDa). (A) Sham operation group, (B) SNCI-induced group, (C) SNCI-induced and 0.1 g/kg SSM-treated group, (D) SNCI-induced and 1 g/kg SSM-treated group, and (E) SNCI-induced and 10 g/kg SSM-treated group. The results are presented as the means ± standard errors of the mean (SEMs). ∗ P
Figure Legend Snippet: Effects of SSM on SNCI-induced protein expression of TNF-α, IL-6, iNOS, and COX-2 . The sciatic nerve was crushed for 30 s using a surgical clip. (a) Relative protein expression levels of TNF- α and IL-6. (b) Relative protein expression levels of iNOS and COX-2. Bands were detected using an enhanced chemiluminescence (ECL) detection kit. Actin was used as an internal control (46 kDa). (A) Sham operation group, (B) SNCI-induced group, (C) SNCI-induced and 0.1 g/kg SSM-treated group, (D) SNCI-induced and 1 g/kg SSM-treated group, and (E) SNCI-induced and 10 g/kg SSM-treated group. The results are presented as the means ± standard errors of the mean (SEMs). ∗ P

Techniques Used: Expressing, Cross-linking Immunoprecipitation

Effects of SSM on LPS-induced protein expression of TNF-α, IL-6, iNOS, and COX-2 . RAW 264.7 cells were induced with 1 μ g/mL LPS and various concentrations of SSM for 24 h. (a) Relative protein expression levels of TNF- α and IL-6. (b) Relative protein expression levels of iNOS and COX-2. Bands were detected using an enhanced chemiluminescence (ECL) detection kit. Actin was used as an internal control (46 kDa). (A) Control group, (B) 1 μ g/mL LPS-administered group, (C) 1 μ g/mL LPS-administered and 0.1 μ g/mL SSM-treated group, (D) 1 μ g/mL LPS-administered and 1 μ g/mL SSM-treated group, and (E) 1 μ g/mL LPS-administered and 10 μ g/mL SSM-treated group. The results are presented as the means ± standard errors of the mean (SEMs). ∗ P
Figure Legend Snippet: Effects of SSM on LPS-induced protein expression of TNF-α, IL-6, iNOS, and COX-2 . RAW 264.7 cells were induced with 1 μ g/mL LPS and various concentrations of SSM for 24 h. (a) Relative protein expression levels of TNF- α and IL-6. (b) Relative protein expression levels of iNOS and COX-2. Bands were detected using an enhanced chemiluminescence (ECL) detection kit. Actin was used as an internal control (46 kDa). (A) Control group, (B) 1 μ g/mL LPS-administered group, (C) 1 μ g/mL LPS-administered and 0.1 μ g/mL SSM-treated group, (D) 1 μ g/mL LPS-administered and 1 μ g/mL SSM-treated group, and (E) 1 μ g/mL LPS-administered and 10 μ g/mL SSM-treated group. The results are presented as the means ± standard errors of the mean (SEMs). ∗ P

Techniques Used: Expressing

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Incubation:

Article Title: Pioglitazone increases VEGFR3 expression and promotes activation of M2 macrophages via the peroxisome proliferator-activated receptor γ
Article Snippet: .. Then they were incubated overnight at 4°C with rabbit anti-arginase1 (Arg1; cat. no. sc-20150; 1:400; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit anti-vascular endothelial growth factor receptor 3 (VEGFR3; cat. no. sc-321; 1:400; Santa Cruz Biotechnology, Inc.), mouse anti-inducible nitric oxide synthase (iNOS; cat. no. sc-7271; 1:200; Santa Cruz Biotechnology, Inc.) and rabbit anti-α-tubulin (1:5,000; cat. no. ab18251; Abcam, Cambridge, MA, USA). .. The membranes containing tissue protein were incubated overnight at 4°C with rabbit anti-α-smooth muscle actin (SMA; cat. no. ab5694; 1:1,000; Abcam), rabbit anti-platelet-derived growth factor receptor (PDGFR)-β (cat. no. ab32570; 1:1,000; Abcam), rabbit anti-PPARγ (cat. no. AP20705a; 1:1,000; Abgent, Inc., San Diego, CA, USA), rabbit anti-phosphorylated (p)-PPARγ (cat. no. ab195925; 1:1,000; Abcam) and mouse anti-GAPDH (1:3,000; Abcam; cat. no. ab8245).

Mouse Assay:

Article Title: Transcriptional and physiological roles for STAT proteins in leptin action
Article Snippet: .. 3.2 STAT1 fails to compensate for the lack of STAT3 in LepRb neurons To determine whether STAT1 might play a role in LepRb signaling and the control of energy balance by leptin, we bred Stat1 flox onto the Lepr cre background to generate Lepr cre/cre ;Stat1 flox/flox (STAT1LepRb KO) mice null for Stat1 in LepRb neurons. .. The deletion of Stat1 from LepRb neurons failed to alter body weight, food intake, adiposity, or glucose homeostasis ( ).

Article Title: Transcriptional and physiological roles for STAT proteins in leptin action
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    Santa Cruz Biotechnology anti inos
    Histological observation of rat hind footpads after injecting Carr 0.9% saline (control group) or Carr. (a-d) H E staining of footpad tissue sections from rat in each group. (e-h) <t>iNOS</t> immunohistochemical staining of footpad tissue sections from rat in each group. (i-l) <t>COX-2</t> immunohistochemical staining of footpad tissue sections from rat in each group. Scale bar = 50 µ m. The infiltrating cells were predominantly neutrophils (N; arrows). The brown staining indicates the interaction of primary and secondary antibodies and the presence of iNOS and COX-2.
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    Histological observation of rat hind footpads after injecting Carr 0.9% saline (control group) or Carr. (a-d) H E staining of footpad tissue sections from rat in each group. (e-h) iNOS immunohistochemical staining of footpad tissue sections from rat in each group. (i-l) COX-2 immunohistochemical staining of footpad tissue sections from rat in each group. Scale bar = 50 µ m. The infiltrating cells were predominantly neutrophils (N; arrows). The brown staining indicates the interaction of primary and secondary antibodies and the presence of iNOS and COX-2.

    Journal: BioMed Research International

    Article Title: Anti-Inflammatory Activities of Leaf Oil from Cinnamomum subavenium In Vitro and In Vivo

    doi: 10.1155/2019/1823149

    Figure Lengend Snippet: Histological observation of rat hind footpads after injecting Carr 0.9% saline (control group) or Carr. (a-d) H E staining of footpad tissue sections from rat in each group. (e-h) iNOS immunohistochemical staining of footpad tissue sections from rat in each group. (i-l) COX-2 immunohistochemical staining of footpad tissue sections from rat in each group. Scale bar = 50 µ m. The infiltrating cells were predominantly neutrophils (N; arrows). The brown staining indicates the interaction of primary and secondary antibodies and the presence of iNOS and COX-2.

    Article Snippet: Anti-iNOS, anti-COX-2 antibody, and anti-NF-κ B p65 antibody were from Santa Cruz Biotechnology (USA).

    Techniques: Staining, Immunohistochemistry

    The effects of steppogenin ( 1 ) on nitrite ( A ) production and iNOS and COX-2 expression ( B ) in lipopolysaccharide (LPS)-stimulated primary rat microglial cells. ( A , B ) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 24 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensities were quantified by densitometry and normalized to the intensities of the β-actin band; the normalized values are presented below each band. ** p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Steppogenin Isolated from Cudrania tricuspidata Shows Antineuroinflammatory Effects via NF-κB and MAPK Pathways in LPS-Stimulated BV2 and Primary Rat Microglial Cells

    doi: 10.3390/molecules22122130

    Figure Lengend Snippet: The effects of steppogenin ( 1 ) on nitrite ( A ) production and iNOS and COX-2 expression ( B ) in lipopolysaccharide (LPS)-stimulated primary rat microglial cells. ( A , B ) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 24 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensities were quantified by densitometry and normalized to the intensities of the β-actin band; the normalized values are presented below each band. ** p

    Article Snippet: Primary antibodies, including mouse/goat/rabbit anti-COX-2 (sc-1745), anti-iNOS (sc-650), anti-β-actin (sc-47778), anti-IкB-α (sc-371), anti-phospho-IкB-α (sc-8404), anti-p50 (sc-7178), anti-p65 (sc-8008), and anti-proliferating cell nuclear antigen (PCNA) (sc-7907), and secondary antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

    Techniques: Expressing

    The effects of steppogenin ( 1 ) on nitrite ( A ) and prostaglandin E2 (PGE 2 ) ( B ) production and iNOS and COX-2 expression ( C ) in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. ( A – C ) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 24 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensity was quantified by densitometry and normalized to the intensity of the β-actin band; the normalized values are presented below each band. * p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Steppogenin Isolated from Cudrania tricuspidata Shows Antineuroinflammatory Effects via NF-κB and MAPK Pathways in LPS-Stimulated BV2 and Primary Rat Microglial Cells

    doi: 10.3390/molecules22122130

    Figure Lengend Snippet: The effects of steppogenin ( 1 ) on nitrite ( A ) and prostaglandin E2 (PGE 2 ) ( B ) production and iNOS and COX-2 expression ( C ) in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. ( A – C ) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 24 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensity was quantified by densitometry and normalized to the intensity of the β-actin band; the normalized values are presented below each band. * p

    Article Snippet: Primary antibodies, including mouse/goat/rabbit anti-COX-2 (sc-1745), anti-iNOS (sc-650), anti-β-actin (sc-47778), anti-IкB-α (sc-371), anti-phospho-IкB-α (sc-8404), anti-p50 (sc-7178), anti-p65 (sc-8008), and anti-proliferating cell nuclear antigen (PCNA) (sc-7907), and secondary antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

    Techniques: Expressing

    Inhibition of iNOS and COX-2 protein expression by scopoletin induced by Carr in mice paw edema for 5th hour. Normal control received 0.9% normal saline. Animals treated with scopoletin (1, 5, and 10 mg/kg) and Indo to injection of Carr right hind paws. The right hind paw tissues were taken at the 5 hour. Then the homogenate was centrifuged and tissue suspended were then prepared and subjected to western blotting using an antibody specific for iNOS and COX-2. β -actin was used as an internal control. (a) Representative western blot from two separate experiments was shown. (b) Relative iNOS and COX-2 protein levels were calculated with reference to Carr-injected mouse. Each point represents the average value for three individual animals . ### compared with sample of control group. The data were presented as mean ± S.E.M. for three different experiments performed in triplicate. ** P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Ameliorative Effects of Scopoletin from Crossostephium chinensis against Inflammation Pain and Its Mechanisms in Mice

    doi: 10.1155/2012/595603

    Figure Lengend Snippet: Inhibition of iNOS and COX-2 protein expression by scopoletin induced by Carr in mice paw edema for 5th hour. Normal control received 0.9% normal saline. Animals treated with scopoletin (1, 5, and 10 mg/kg) and Indo to injection of Carr right hind paws. The right hind paw tissues were taken at the 5 hour. Then the homogenate was centrifuged and tissue suspended were then prepared and subjected to western blotting using an antibody specific for iNOS and COX-2. β -actin was used as an internal control. (a) Representative western blot from two separate experiments was shown. (b) Relative iNOS and COX-2 protein levels were calculated with reference to Carr-injected mouse. Each point represents the average value for three individual animals . ### compared with sample of control group. The data were presented as mean ± S.E.M. for three different experiments performed in triplicate. ** P

    Article Snippet: Anti-iNOS, anti-COX-2, and anti-β -actin antibody (Santa Cruz, USA), and a protein assay kit (Bio-Rad Laboratories Ltd., Watford, Hertfordshire, UK) were obtained as indicated.

    Techniques: Inhibition, Expressing, Mouse Assay, Injection, Western Blot

    CAMK4 is necessary for crocin-mediated inhibition of iNOS expression in LPS-stimulated macrophages. RAW 264.7 cells were transfected with CAMK4 siRNA or control siRNA and then treated with crocin (500 μ M). After 3 h, cells were incubated with LPS (0.1 μ g/mL) for 24 h. Equal amounts of cytosolic extract were analyzed by Western blotting. Tubulin was used as a loading control.

    Journal: Mediators of Inflammation

    Article Title: Crocin Suppresses LPS-Stimulated Expression of Inducible Nitric Oxide Synthase by Upregulation of Heme Oxygenase-1 via Calcium/Calmodulin-Dependent Protein Kinase 4

    doi: 10.1155/2014/728709

    Figure Lengend Snippet: CAMK4 is necessary for crocin-mediated inhibition of iNOS expression in LPS-stimulated macrophages. RAW 264.7 cells were transfected with CAMK4 siRNA or control siRNA and then treated with crocin (500 μ M). After 3 h, cells were incubated with LPS (0.1 μ g/mL) for 24 h. Equal amounts of cytosolic extract were analyzed by Western blotting. Tubulin was used as a loading control.

    Article Snippet: The gel was then transferred to 0.45 μ m nitrocellulose paper and incubated with anti-iNOS, p65, HO-1, Nrf2, phospho-CAMK4, Akt, ERK1/2, JNK, TBP, HDAC antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CAMK4, phospho-Akt, phospho-ERK1/2, phospho-JNK antibodies (Cell Signaling Technology, Beverly, MA, USA) or α -tubulin antibody (Bio Genex, Fremont, CA, USA), and secondary antibody and then detected by an enhanced chemiluminescence detection system according to the recommended procedure (GE Healthcare, Piscataway, NJ, USA).

    Techniques: Inhibition, Expressing, Transfection, Incubation, Western Blot