Structured Review

Santa Cruz Biotechnology anti inos
Celastrol inhibits oxLDL induced NO production and inflammatory gene expression in RAW 264.7 cells. RAW 264.7 cells were exposed to oxLDL (80 µg/mL) in the presence or absence of celastrol (50–200 nM) for 24 h. Western blot analysis and quantification of <t>iNOS</t> protein expression (A). The iNOS inhibitor 1400w (200 µmol/L) and oxLDL (80 µg/mL) were incubated with RAW 264.7 cells for 24 hours. Representative photographs showing RAW 264.7 cells stained with oil red O (B). Quantification of NO, IL-6, <t>TNF-α</t> (carried out by real-time PCR )(C–E). ## p
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1) Product Images from "Celastrol Prevents Atherosclerosis via Inhibiting LOX-1 and Oxidative Stress"

Article Title: Celastrol Prevents Atherosclerosis via Inhibiting LOX-1 and Oxidative Stress

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065477

Celastrol inhibits oxLDL induced NO production and inflammatory gene expression in RAW 264.7 cells. RAW 264.7 cells were exposed to oxLDL (80 µg/mL) in the presence or absence of celastrol (50–200 nM) for 24 h. Western blot analysis and quantification of iNOS protein expression (A). The iNOS inhibitor 1400w (200 µmol/L) and oxLDL (80 µg/mL) were incubated with RAW 264.7 cells for 24 hours. Representative photographs showing RAW 264.7 cells stained with oil red O (B). Quantification of NO, IL-6, TNF-α (carried out by real-time PCR )(C–E). ## p
Figure Legend Snippet: Celastrol inhibits oxLDL induced NO production and inflammatory gene expression in RAW 264.7 cells. RAW 264.7 cells were exposed to oxLDL (80 µg/mL) in the presence or absence of celastrol (50–200 nM) for 24 h. Western blot analysis and quantification of iNOS protein expression (A). The iNOS inhibitor 1400w (200 µmol/L) and oxLDL (80 µg/mL) were incubated with RAW 264.7 cells for 24 hours. Representative photographs showing RAW 264.7 cells stained with oil red O (B). Quantification of NO, IL-6, TNF-α (carried out by real-time PCR )(C–E). ## p

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

2) Product Images from "Microglia and motor neurons during disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis: changes in arginase1 and inducible nitric oxide synthase"

Article Title: Microglia and motor neurons during disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis: changes in arginase1 and inducible nitric oxide synthase

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-11-55

Expression of arginase1 (Arg1) and inducible nitric oxide synthase (iNOS) in microglia of the ventral horn of wild-type (WT) and amyotrophic lateral sclerosis-linked mutant human superoxide dismutase transgene (SOD1 G93A ) cervical spinal cord. The number of Arg1-negative and Arg1-positive cervical microglia remained relatively constant over time in WT mice (A) . In the cervical spinal cord of SOD1 G93A mice, the number of Arg1-positive and Arg1-negative microglia remained equivalent until 22 weeks of age, when there was an increase in the number of Arg1-positive microglia (B) . There were greater numbers of iNOS-negative microglia than iNOS-positive microglia throughout the time course in WT mice (C) . The numbers of iNOS-positive microglia and iNOS-negative microglia both increased with disease progression in the SOD1 G93A cervical spinal cord (D) . * P
Figure Legend Snippet: Expression of arginase1 (Arg1) and inducible nitric oxide synthase (iNOS) in microglia of the ventral horn of wild-type (WT) and amyotrophic lateral sclerosis-linked mutant human superoxide dismutase transgene (SOD1 G93A ) cervical spinal cord. The number of Arg1-negative and Arg1-positive cervical microglia remained relatively constant over time in WT mice (A) . In the cervical spinal cord of SOD1 G93A mice, the number of Arg1-positive and Arg1-negative microglia remained equivalent until 22 weeks of age, when there was an increase in the number of Arg1-positive microglia (B) . There were greater numbers of iNOS-negative microglia than iNOS-positive microglia throughout the time course in WT mice (C) . The numbers of iNOS-positive microglia and iNOS-negative microglia both increased with disease progression in the SOD1 G93A cervical spinal cord (D) . * P

Techniques Used: Expressing, Mutagenesis, Mouse Assay

Inducible nitric oxide synthase expression in microglia of amyotrophic lateral sclerosis-linked mutant human superoxide dismutase transgene (SOD1 G93A ) and wild-type (WT) lumbar spinal cord ventral horn. The majority of tomato lectin (TL)-positive microglia (red) in the ventral horn did not express inducible nitric oxide synthase (iNOS) (arrows, A,B) at 6 weeks of age in either WT (A) or SOD1 G93A (B) mice. A subset of microglia were iNOS-positive (green) (arrows, C,D). The numbers of iNOS-positive and iNOS-negative microglia stayed relatively constant over time in WT mice (E) . The number of iNOS-positive microglia increased with disease progression in SOD1 G93A mice; the number of iNOS-negative microglia increased at 22 and 25 weeks of age in SOD1 G93A mice (F) . The combined data from Figure 2 F and 3 F show that the number of both arginase1 (Arg1)-positive and iNOS-positive microglia increased with disease progression (G) ; the percentage of microglia expressing Arg1 and expressing iNOS also increased with time (H) . Scale bar 50 μm in A-D. * P
Figure Legend Snippet: Inducible nitric oxide synthase expression in microglia of amyotrophic lateral sclerosis-linked mutant human superoxide dismutase transgene (SOD1 G93A ) and wild-type (WT) lumbar spinal cord ventral horn. The majority of tomato lectin (TL)-positive microglia (red) in the ventral horn did not express inducible nitric oxide synthase (iNOS) (arrows, A,B) at 6 weeks of age in either WT (A) or SOD1 G93A (B) mice. A subset of microglia were iNOS-positive (green) (arrows, C,D). The numbers of iNOS-positive and iNOS-negative microglia stayed relatively constant over time in WT mice (E) . The number of iNOS-positive microglia increased with disease progression in SOD1 G93A mice; the number of iNOS-negative microglia increased at 22 and 25 weeks of age in SOD1 G93A mice (F) . The combined data from Figure 2 F and 3 F show that the number of both arginase1 (Arg1)-positive and iNOS-positive microglia increased with disease progression (G) ; the percentage of microglia expressing Arg1 and expressing iNOS also increased with time (H) . Scale bar 50 μm in A-D. * P

Techniques Used: Expressing, Mutagenesis, Mouse Assay

Percentage area occupied by arginase1 (Arg1) and inducible nitric oxide synthase (iNOS) immunoreactivity in amyotrophic lateral sclerosis-linked mutant human superoxide dismutase transgene (SOD1 G93A ) lumbar spinal cord microglia. The percentage area of the microglial cell body occupied by Arg1 (A,B) or iNOS (C,D) positive immunoreactivity was examined at 6 weeks (A,C) and 22 weeks (B,D) of age in a subset of microglia displaying the strongest immunoreactivity. The percentage of cell body area occupied by Arg1 was higher at 22 weeks of age than at 6 weeks of age; the percentage of cell body area occupied by iNOS was also higher at 22 weeks of age than at 6 weeks of age (E) . Scale bar 10 μm in A-D. *** P
Figure Legend Snippet: Percentage area occupied by arginase1 (Arg1) and inducible nitric oxide synthase (iNOS) immunoreactivity in amyotrophic lateral sclerosis-linked mutant human superoxide dismutase transgene (SOD1 G93A ) lumbar spinal cord microglia. The percentage area of the microglial cell body occupied by Arg1 (A,B) or iNOS (C,D) positive immunoreactivity was examined at 6 weeks (A,C) and 22 weeks (B,D) of age in a subset of microglia displaying the strongest immunoreactivity. The percentage of cell body area occupied by Arg1 was higher at 22 weeks of age than at 6 weeks of age; the percentage of cell body area occupied by iNOS was also higher at 22 weeks of age than at 6 weeks of age (E) . Scale bar 10 μm in A-D. *** P

Techniques Used: Mutagenesis

3) Product Images from "Ghrelin protects against contact dermatitis and psoriasiform skin inflammation by antagonizing TNF-α/NF-κB signaling pathways"

Article Title: Ghrelin protects against contact dermatitis and psoriasiform skin inflammation by antagonizing TNF-α/NF-κB signaling pathways

Journal: Scientific Reports

doi: 10.1038/s41598-018-38174-2

Ghrelin improves psoriasis in an IMQ-induced psoriasis mouse model. ( A ) Improved skin conditions were observed in mice treated with ghrelin. ( B ) Ghrelin treatment led to reduced PASI scores in an IMQ-treated psoriasis mouse model. ( C , D ) A smoother epidermis, lesser degree of parakeratosis, and reduced epidermal thickening were demonstrated upon treatment with ghrelin, as shown by both HE staining and the measurement of epithelial thickness. ( E ) Ghrelin downregulates the expression of CD68, supporting the anti-inflammatory effect of ghrelin in IMQ-induced psoriasis, as shown by immunohistochemistry. ( F ) iNOS is decreased in response to treatment with ghrelin, as shown by immunohistochemistry. ( G , H ) Total protein was extracted from skin tissue, and iNOS expression was decreased upon treatment with ghrelin, as shown by western blotting. ( I – K ) Ghrelin application significantly attenuated the mRNA expression of IL-1β, IL-6 and iNOS, as shown by real-time PCR. ( L , M ) The serum levels of IL-1β and IL-6 were notably reduced by ghrelin administration, as shown by ELISA. (*p
Figure Legend Snippet: Ghrelin improves psoriasis in an IMQ-induced psoriasis mouse model. ( A ) Improved skin conditions were observed in mice treated with ghrelin. ( B ) Ghrelin treatment led to reduced PASI scores in an IMQ-treated psoriasis mouse model. ( C , D ) A smoother epidermis, lesser degree of parakeratosis, and reduced epidermal thickening were demonstrated upon treatment with ghrelin, as shown by both HE staining and the measurement of epithelial thickness. ( E ) Ghrelin downregulates the expression of CD68, supporting the anti-inflammatory effect of ghrelin in IMQ-induced psoriasis, as shown by immunohistochemistry. ( F ) iNOS is decreased in response to treatment with ghrelin, as shown by immunohistochemistry. ( G , H ) Total protein was extracted from skin tissue, and iNOS expression was decreased upon treatment with ghrelin, as shown by western blotting. ( I – K ) Ghrelin application significantly attenuated the mRNA expression of IL-1β, IL-6 and iNOS, as shown by real-time PCR. ( L , M ) The serum levels of IL-1β and IL-6 were notably reduced by ghrelin administration, as shown by ELISA. (*p

Techniques Used: Mouse Assay, Staining, Expressing, Immunohistochemistry, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Ghrelin attenuates the secretion of proinflammatory cytokines in an OXA-induced contact dermatitis mouse model. ( A – F ) The levels of IL-1β, IL-6, iNOS, COX-2, TNF-α and MMP-3 were dramatically decreased upon treatment with ghrelin, as shown by real-time PCR. ( G ) iNOS expression was downregulated in mice treated with ghrelin in OXA-induced contact dermatitis, as shown by immunohistochemistry. ( H , I ) The protein levels of iNOS and COX-2 were markedly inhibited with ghrelin treatment, as shown by western blotting. ( J , K ) IL-1β and IL-6 were detected in mouse serum, indicating the ghrelin-mediated reduction in inflammatory cytokines in serum, as shown by ELISA. (*p
Figure Legend Snippet: Ghrelin attenuates the secretion of proinflammatory cytokines in an OXA-induced contact dermatitis mouse model. ( A – F ) The levels of IL-1β, IL-6, iNOS, COX-2, TNF-α and MMP-3 were dramatically decreased upon treatment with ghrelin, as shown by real-time PCR. ( G ) iNOS expression was downregulated in mice treated with ghrelin in OXA-induced contact dermatitis, as shown by immunohistochemistry. ( H , I ) The protein levels of iNOS and COX-2 were markedly inhibited with ghrelin treatment, as shown by western blotting. ( J , K ) IL-1β and IL-6 were detected in mouse serum, indicating the ghrelin-mediated reduction in inflammatory cytokines in serum, as shown by ELISA. (*p

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Mouse Assay, Immunohistochemistry, Western Blot, Enzyme-linked Immunosorbent Assay

Ghrelin inhibits inflammatory reactions by combining with GHSR1a and antagonizing TNF-α signaling. ( A – C ) Ghrelin greatly inhibited the secretion of inflammatory cytokines, such as IL-1β, IL-6, and iNOS, in vitro in a dose-dependent manner, as shown by real-time PCR. ( D ) Reduced nitrite levels were promoted by the application of ghrelin in a dose-dependent manner, as shown by the Griess experiment. ( E , F ) iNOS expression is downregulated in response to ghrelin application in a dose-dependent manner, as shown by western blotting. ( G , H ) IL-1β and IL-6 secretion were changed by ghrelin pretreatment and appeared to be heightened in proportion to elevations in ghrelin concentration, as shown by ELISA. ( I – K ) The mRNA expression of IL-1β, IL-6 and iNOS was enhanced by application of DLys, implying that DLys antagonizes the effect of ghrelin, as shown by real-time PCR. ( L ) Addition of DLys attenuates ghrelin’s effect of decreasing nitrite production, as detected by the Griess test. ( M , N ) Higher iNOS protein levels occurred with DLys application, as shown by western blotting. ( O , P ) Application of DLys inhibited ghrelin’s antagonizing effect on the secretion of inflammatory cytokines IL-1β and IL-6 induced by stimulation with TNF-α, as shown by ELISA. (*p
Figure Legend Snippet: Ghrelin inhibits inflammatory reactions by combining with GHSR1a and antagonizing TNF-α signaling. ( A – C ) Ghrelin greatly inhibited the secretion of inflammatory cytokines, such as IL-1β, IL-6, and iNOS, in vitro in a dose-dependent manner, as shown by real-time PCR. ( D ) Reduced nitrite levels were promoted by the application of ghrelin in a dose-dependent manner, as shown by the Griess experiment. ( E , F ) iNOS expression is downregulated in response to ghrelin application in a dose-dependent manner, as shown by western blotting. ( G , H ) IL-1β and IL-6 secretion were changed by ghrelin pretreatment and appeared to be heightened in proportion to elevations in ghrelin concentration, as shown by ELISA. ( I – K ) The mRNA expression of IL-1β, IL-6 and iNOS was enhanced by application of DLys, implying that DLys antagonizes the effect of ghrelin, as shown by real-time PCR. ( L ) Addition of DLys attenuates ghrelin’s effect of decreasing nitrite production, as detected by the Griess test. ( M , N ) Higher iNOS protein levels occurred with DLys application, as shown by western blotting. ( O , P ) Application of DLys inhibited ghrelin’s antagonizing effect on the secretion of inflammatory cytokines IL-1β and IL-6 induced by stimulation with TNF-α, as shown by ELISA. (*p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

4) Product Images from "Ginkgolide C Alleviates Myocardial Ischemia/Reperfusion-Induced Inflammatory Injury via Inhibition of CD40-NF-κB Pathway"

Article Title: Ginkgolide C Alleviates Myocardial Ischemia/Reperfusion-Induced Inflammatory Injury via Inhibition of CD40-NF-κB Pathway

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00109

Effects of GC on cell viability (A) and the expressions of (B) CD40, (C) ICAM-1, (D) VCAM-1, (E) iNOS, (F) cytoplasm NF-κB p65, (G) nucleus NF-κB p65, (H) p-IκB-α and (I) IKK-β by Western blot after CD40 silencing procedure. Results were expressed as Protein/reference protein ratio. Data were expressed as mean ± SD of three independent experiments. ## P
Figure Legend Snippet: Effects of GC on cell viability (A) and the expressions of (B) CD40, (C) ICAM-1, (D) VCAM-1, (E) iNOS, (F) cytoplasm NF-κB p65, (G) nucleus NF-κB p65, (H) p-IκB-α and (I) IKK-β by Western blot after CD40 silencing procedure. Results were expressed as Protein/reference protein ratio. Data were expressed as mean ± SD of three independent experiments. ## P

Techniques Used: Western Blot

Effect of GC on ICAM-1, VCAM-1 and iNOS expressions in myocardial tissue after I/R procedure. The tissue was observed using a microscope at a magnification × 400. (A) GC decreased the expression of ICAM-1. (B) GC decreased the expression of VCAM-1. (C) GC decreased the expression of iNOS. There was a little expression of ICAM-1, VCAM-1 and iNOS in myocardial tissue of the control group. The expressions of ICAM-1, VCAM-1 and iNOS in I/R group were markedly increased. Administration of GC exhibited reduced expressions of ICAM-1, VCAM-1 and iNOS compared with the I/R group in a dose-dependent manner. Administration of Aspirin also significantly decreased the expressions of ICAM-1, VCAM-1 and iNOS compared with I/R group. The location of the histological images was taken in the infarcted area. Data were expressed as mean ± SD ( n = 8). ## P
Figure Legend Snippet: Effect of GC on ICAM-1, VCAM-1 and iNOS expressions in myocardial tissue after I/R procedure. The tissue was observed using a microscope at a magnification × 400. (A) GC decreased the expression of ICAM-1. (B) GC decreased the expression of VCAM-1. (C) GC decreased the expression of iNOS. There was a little expression of ICAM-1, VCAM-1 and iNOS in myocardial tissue of the control group. The expressions of ICAM-1, VCAM-1 and iNOS in I/R group were markedly increased. Administration of GC exhibited reduced expressions of ICAM-1, VCAM-1 and iNOS compared with the I/R group in a dose-dependent manner. Administration of Aspirin also significantly decreased the expressions of ICAM-1, VCAM-1 and iNOS compared with I/R group. The location of the histological images was taken in the infarcted area. Data were expressed as mean ± SD ( n = 8). ## P

Techniques Used: Microscopy, Expressing

Effects of GC on the expressions of CD40, ICAM-1, VCAM-1, iNOS, NF-κB p65, p-IκB-α and IKK-β by Western blot after H/R procedure. (A) GC decreased the expression of CD40. (B) GC decreased the expression of ICAM-1. (C) GC decreased the expression of VCAM-1. (D) GC decreased the expression of iNOS. GC blocked the translocation of NF-κB p65 from cytosolic (E) to nuclear (F) . (G) GC down-regulated the expression of p-IκB-α. (H) GC decreased the expression of IKK-β. CD40, ICAM-1, VCAM-1, iNOS, p-IκB-α and IKK-β proteins were measured in cytosolic extract. The NF-κB p65 protein levels were assayed separately in cytosolic and nuclear extracts. Results were expressed as Protein/reference protein ratio. Data were expressed as mean ± SD of three independent experiments. ## P
Figure Legend Snippet: Effects of GC on the expressions of CD40, ICAM-1, VCAM-1, iNOS, NF-κB p65, p-IκB-α and IKK-β by Western blot after H/R procedure. (A) GC decreased the expression of CD40. (B) GC decreased the expression of ICAM-1. (C) GC decreased the expression of VCAM-1. (D) GC decreased the expression of iNOS. GC blocked the translocation of NF-κB p65 from cytosolic (E) to nuclear (F) . (G) GC down-regulated the expression of p-IκB-α. (H) GC decreased the expression of IKK-β. CD40, ICAM-1, VCAM-1, iNOS, p-IκB-α and IKK-β proteins were measured in cytosolic extract. The NF-κB p65 protein levels were assayed separately in cytosolic and nuclear extracts. Results were expressed as Protein/reference protein ratio. Data were expressed as mean ± SD of three independent experiments. ## P

Techniques Used: Western Blot, Expressing, Translocation Assay

5) Product Images from "Manganese Activates NLRP3 Inflammasome Signaling and Propagates Exosomal Release of ASC in Microglial Cells"

Article Title: Manganese Activates NLRP3 Inflammasome Signaling and Propagates Exosomal Release of ASC in Microglial Cells

Journal: Science signaling

doi: 10.1126/scisignal.aat9900

Manganese exposure induced NLRP3 inflammasome activation in microglial cells in vivo ( A ) Western Blot analysis of the NLRP3 and iNOS expression in wild-type microglial cells treated with Mn and αSyn Agg as indicated. Blots (left) are representative of 3 independent experiments. Normalized band intensity data (right) are means ± SEM from all experiments. ( B ) Luminex analysis of IL-1β production by wild-type microglial cells treated with Mn and αSyn Agg as indicated. Data are means ± SEM pooled from 4 independent experiments. ( C ) qRT-PCR analysis of NLRP3, NLRC4, and AIM2 mRNA expression in the striata of C57BL mice exposed to Mn for 30 days. Data are means ± SEM pooled from 5 mice from 3 experiments. ( D ) Western blot analysis of Caspase 1 cleavage and IL-1β maturation in lysates from striatum samples from mice treated as indicated. Blots (upper) are representative of 6 mice from 3 experiments. Normalized band intensity data (lower) are means ± SEM from all experiments. ( E ) Immunofluorescence microscopy analysis of NLRP3 abundance in IBA1-positive microglial cells in the striatal region of mice treated as indicated. Images are representative of 3 mice from 3 experiments. Scale bar, 15 μm. *P
Figure Legend Snippet: Manganese exposure induced NLRP3 inflammasome activation in microglial cells in vivo ( A ) Western Blot analysis of the NLRP3 and iNOS expression in wild-type microglial cells treated with Mn and αSyn Agg as indicated. Blots (left) are representative of 3 independent experiments. Normalized band intensity data (right) are means ± SEM from all experiments. ( B ) Luminex analysis of IL-1β production by wild-type microglial cells treated with Mn and αSyn Agg as indicated. Data are means ± SEM pooled from 4 independent experiments. ( C ) qRT-PCR analysis of NLRP3, NLRC4, and AIM2 mRNA expression in the striata of C57BL mice exposed to Mn for 30 days. Data are means ± SEM pooled from 5 mice from 3 experiments. ( D ) Western blot analysis of Caspase 1 cleavage and IL-1β maturation in lysates from striatum samples from mice treated as indicated. Blots (upper) are representative of 6 mice from 3 experiments. Normalized band intensity data (lower) are means ± SEM from all experiments. ( E ) Immunofluorescence microscopy analysis of NLRP3 abundance in IBA1-positive microglial cells in the striatal region of mice treated as indicated. Images are representative of 3 mice from 3 experiments. Scale bar, 15 μm. *P

Techniques Used: Activation Assay, In Vivo, Western Blot, Expressing, Luminex, Quantitative RT-PCR, Mouse Assay, Immunofluorescence, Microscopy

6) Product Images from "Ameliorative effect of taurine-chloramine in azathioprine-induced testicular damage; a deeper insight into the mechanism of protection"

Article Title: Ameliorative effect of taurine-chloramine in azathioprine-induced testicular damage; a deeper insight into the mechanism of protection

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/s12906-018-2272-z

Western blotting analysis of testicular inducible nitric oxide synthase ( a ; iNOS) and p65 nuclear factor-kappa B ( b ; NF-ƘB p65) in Control, Taurine-chloramine (TAU-CL), Azathioprine (AZA) and TAU-CL/AZA groups. Data are presented as mean ± SEM, a Significant change from the control group at p
Figure Legend Snippet: Western blotting analysis of testicular inducible nitric oxide synthase ( a ; iNOS) and p65 nuclear factor-kappa B ( b ; NF-ƘB p65) in Control, Taurine-chloramine (TAU-CL), Azathioprine (AZA) and TAU-CL/AZA groups. Data are presented as mean ± SEM, a Significant change from the control group at p

Techniques Used: Western Blot

7) Product Images from "Neuroprotective Effect of Artesunate in Experimental Model of Traumatic Brain Injury"

Article Title: Neuroprotective Effect of Artesunate in Experimental Model of Traumatic Brain Injury

Journal: Frontiers in Neurology

doi: 10.3389/fneur.2018.00590

Protective effect of artesunate treatment on cytokines and iNOS expression. (B,F) Showed a significant increase of expression for IL-1β and TNF-α respectively, 24 h after TBI injury compared with sham group (A,E) . When compared with vehicle group, brain section from mice treated with artesunate show a significant reduction in IL-1β and TNF-α respectively (C,G) , see densitometric analysis (D) for IL-1β, and (H) for TNF-α. The immunohistochemical analysis for iNOS, show that 24 h after TBI injury in the vehicle group there is a significant increase in iNOS expression as show in (J) compared with sham group (I) . While as show in (K) treatment with artesunate significantly reduce the iNOS expression compared to vehicle group, see densitometric analysis (L) . Data are expressed as Mean ± SEM from N = 10 Mice for each group. *** P
Figure Legend Snippet: Protective effect of artesunate treatment on cytokines and iNOS expression. (B,F) Showed a significant increase of expression for IL-1β and TNF-α respectively, 24 h after TBI injury compared with sham group (A,E) . When compared with vehicle group, brain section from mice treated with artesunate show a significant reduction in IL-1β and TNF-α respectively (C,G) , see densitometric analysis (D) for IL-1β, and (H) for TNF-α. The immunohistochemical analysis for iNOS, show that 24 h after TBI injury in the vehicle group there is a significant increase in iNOS expression as show in (J) compared with sham group (I) . While as show in (K) treatment with artesunate significantly reduce the iNOS expression compared to vehicle group, see densitometric analysis (L) . Data are expressed as Mean ± SEM from N = 10 Mice for each group. *** P

Techniques Used: Expressing, Mouse Assay, Immunohistochemistry

8) Product Images from "Cell permeable HMGB1-binding heptamer peptide ameliorates neurovascular complications associated with thrombolytic therapy in rats with transient ischemic stroke"

Article Title: Cell permeable HMGB1-binding heptamer peptide ameliorates neurovascular complications associated with thrombolytic therapy in rats with transient ischemic stroke

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-018-1267-5

HBHP alleviated inflammatory reactions in tPA-treated stroke rats. a ELISA data of serum IL-1β levels in stroke rats at 8 h after ischemia, n = 5–7. b Quantitative real-time PCR analysis of mRNA expressions of IL-1β, IL-6, IL-12b, and TNF-α in the peri-infarct cortex from 4.5 h MCAO rats or in the same regions of sham-operated rats, n = 3–4. c Western blot and quantified data of iNOS, COX-2, and IL-1β in ischemic ipsilateral hemispheres at 8 h after ischemia, n = 3–4. NS: not significant; * P
Figure Legend Snippet: HBHP alleviated inflammatory reactions in tPA-treated stroke rats. a ELISA data of serum IL-1β levels in stroke rats at 8 h after ischemia, n = 5–7. b Quantitative real-time PCR analysis of mRNA expressions of IL-1β, IL-6, IL-12b, and TNF-α in the peri-infarct cortex from 4.5 h MCAO rats or in the same regions of sham-operated rats, n = 3–4. c Western blot and quantified data of iNOS, COX-2, and IL-1β in ischemic ipsilateral hemispheres at 8 h after ischemia, n = 3–4. NS: not significant; * P

Techniques Used: Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot

9) Product Images from "High glucose enhances LPS-stimulated human PMVEC hyperpermeability via the NO pathway"

Article Title: High glucose enhances LPS-stimulated human PMVEC hyperpermeability via the NO pathway

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2013.1166

Effect of lipopolysaccharide (LPS) and a high glucose concentration on expression of dimethylarginine dimethylaminohydrolase (DDAH)-2, inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in cultured human pulmonary microvascular endothelial cells (PMVECs). Cells were incubated with normal (5.5 mM) or high (33 mM) D-glucose concentrations for 5 days in medium with 2% serum (to maintain the cells in the quiescent state), and then incubated with 10 μ g/ml LPS for 12 h. Cellular protein was isolated, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to membranes for western blotting as described in Materials and methods (A). Densitometry was performed to quantify the expression (B–D). NG, normal glucose group; NG + LPS, normal glucose + LPS group; HG, high glucose group; HG + LPS, high glucose + LPS group. Data are expressed as mean ± standard deviation (SD; n=5). * P
Figure Legend Snippet: Effect of lipopolysaccharide (LPS) and a high glucose concentration on expression of dimethylarginine dimethylaminohydrolase (DDAH)-2, inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in cultured human pulmonary microvascular endothelial cells (PMVECs). Cells were incubated with normal (5.5 mM) or high (33 mM) D-glucose concentrations for 5 days in medium with 2% serum (to maintain the cells in the quiescent state), and then incubated with 10 μ g/ml LPS for 12 h. Cellular protein was isolated, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to membranes for western blotting as described in Materials and methods (A). Densitometry was performed to quantify the expression (B–D). NG, normal glucose group; NG + LPS, normal glucose + LPS group; HG, high glucose group; HG + LPS, high glucose + LPS group. Data are expressed as mean ± standard deviation (SD; n=5). * P

Techniques Used: Concentration Assay, Expressing, Cell Culture, Incubation, Isolation, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Standard Deviation

10) Product Images from "Ginkgolide C Alleviates Myocardial Ischemia/Reperfusion-Induced Inflammatory Injury via Inhibition of CD40-NF-κB Pathway"

Article Title: Ginkgolide C Alleviates Myocardial Ischemia/Reperfusion-Induced Inflammatory Injury via Inhibition of CD40-NF-κB Pathway

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00109

Effect of GC on ICAM-1, VCAM-1 and iNOS expressions in myocardial tissue after I/R procedure. The tissue was observed using a microscope at a magnification × 400. (A) GC decreased the expression of ICAM-1. (B) GC decreased the expression of VCAM-1. (C) GC decreased the expression of iNOS. There was a little expression of ICAM-1, VCAM-1 and iNOS in myocardial tissue of the control group. The expressions of ICAM-1, VCAM-1 and iNOS in I/R group were markedly increased. Administration of GC exhibited reduced expressions of ICAM-1, VCAM-1 and iNOS compared with the I/R group in a dose-dependent manner. Administration of Aspirin also significantly decreased the expressions of ICAM-1, VCAM-1 and iNOS compared with I/R group. The location of the histological images was taken in the infarcted area. Data were expressed as mean ± SD ( n = 8). ## P
Figure Legend Snippet: Effect of GC on ICAM-1, VCAM-1 and iNOS expressions in myocardial tissue after I/R procedure. The tissue was observed using a microscope at a magnification × 400. (A) GC decreased the expression of ICAM-1. (B) GC decreased the expression of VCAM-1. (C) GC decreased the expression of iNOS. There was a little expression of ICAM-1, VCAM-1 and iNOS in myocardial tissue of the control group. The expressions of ICAM-1, VCAM-1 and iNOS in I/R group were markedly increased. Administration of GC exhibited reduced expressions of ICAM-1, VCAM-1 and iNOS compared with the I/R group in a dose-dependent manner. Administration of Aspirin also significantly decreased the expressions of ICAM-1, VCAM-1 and iNOS compared with I/R group. The location of the histological images was taken in the infarcted area. Data were expressed as mean ± SD ( n = 8). ## P

Techniques Used: Microscopy, Expressing

Effects of GC on the expressions of CD40, ICAM-1, VCAM-1, iNOS, NF-κB p65, p-IκB-α and IKK-β by Western blot after H/R procedure. (A) GC decreased the expression of CD40. (B) GC decreased the expression of ICAM-1. (C) GC decreased the expression of VCAM-1. (D) GC decreased the expression of iNOS. GC blocked the translocation of NF-κB p65 from cytosolic (E) to nuclear (F) . (G) GC down-regulated the expression of p-IκB-α. (H) GC decreased the expression of IKK-β. CD40, ICAM-1, VCAM-1, iNOS, p-IκB-α and IKK-β proteins were measured in cytosolic extract. The NF-κB p65 protein levels were assayed separately in cytosolic and nuclear extracts. Results were expressed as Protein/reference protein ratio. Data were expressed as mean ± SD of three independent experiments. ## P
Figure Legend Snippet: Effects of GC on the expressions of CD40, ICAM-1, VCAM-1, iNOS, NF-κB p65, p-IκB-α and IKK-β by Western blot after H/R procedure. (A) GC decreased the expression of CD40. (B) GC decreased the expression of ICAM-1. (C) GC decreased the expression of VCAM-1. (D) GC decreased the expression of iNOS. GC blocked the translocation of NF-κB p65 from cytosolic (E) to nuclear (F) . (G) GC down-regulated the expression of p-IκB-α. (H) GC decreased the expression of IKK-β. CD40, ICAM-1, VCAM-1, iNOS, p-IκB-α and IKK-β proteins were measured in cytosolic extract. The NF-κB p65 protein levels were assayed separately in cytosolic and nuclear extracts. Results were expressed as Protein/reference protein ratio. Data were expressed as mean ± SD of three independent experiments. ## P

Techniques Used: Western Blot, Expressing, Translocation Assay

11) Product Images from "Isolation of Novel Sesquiterpeniods and Anti-neuroinflammatory Metabolites from Nardostachys jatamansi"

Article Title: Isolation of Novel Sesquiterpeniods and Anti-neuroinflammatory Metabolites from Nardostachys jatamansi

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

doi: 10.3390/molecules23092367

The effects of compounds ( A ) 3 , ( B ) 4 , and ( C ) 8 on the expression of iNOS and COX-2 proteins in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. (A–C) The cells were pretreated for 3 h with the indicated concentrations of compounds 3 , 4 , and 8, and then stimulated for 24 h with LPS (1.0 μg/mL). The data are presented as the means ± SD of three experiments. The band intensity was quantified by densitometry and normalized to the intensity of the β-actin band; lower panel, summarized bar graphs show band intensity presented as ratio of targeting protein over β-actin. The significance of the comparison against the LPS-treated group is indicated as follows: *** p
Figure Legend Snippet: The effects of compounds ( A ) 3 , ( B ) 4 , and ( C ) 8 on the expression of iNOS and COX-2 proteins in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. (A–C) The cells were pretreated for 3 h with the indicated concentrations of compounds 3 , 4 , and 8, and then stimulated for 24 h with LPS (1.0 μg/mL). The data are presented as the means ± SD of three experiments. The band intensity was quantified by densitometry and normalized to the intensity of the β-actin band; lower panel, summarized bar graphs show band intensity presented as ratio of targeting protein over β-actin. The significance of the comparison against the LPS-treated group is indicated as follows: *** p

Techniques Used: Expressing

12) Product Images from "12/15-Lipoxygenase metabolites of arachidonic acid activate PPARγ: a possible neuroprotective effect in ischemic brain"

Article Title: 12/15-Lipoxygenase metabolites of arachidonic acid activate PPARγ: a possible neuroprotective effect in ischemic brain

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M053058

12(S)-HETE and 15(S)-HETE inhibited the induction of COX-2, iNOS, and NF-κB in ischemic brain. Focal ischemia was induced by transient MCAO. 12(S)-HETE or 15(S)-HETE (10, 15, and 20 μg) or vehicle was injected icv 30 min before the onset of MCAO. The COX-2 and iNOS protein levels, NO content, and NF-κB activation in ipsilateral (ischemic) cortex were estimated at 24 h post-MCAO. Data are expressed as mean ± SEM; n = 8/group; * P
Figure Legend Snippet: 12(S)-HETE and 15(S)-HETE inhibited the induction of COX-2, iNOS, and NF-κB in ischemic brain. Focal ischemia was induced by transient MCAO. 12(S)-HETE or 15(S)-HETE (10, 15, and 20 μg) or vehicle was injected icv 30 min before the onset of MCAO. The COX-2 and iNOS protein levels, NO content, and NF-κB activation in ipsilateral (ischemic) cortex were estimated at 24 h post-MCAO. Data are expressed as mean ± SEM; n = 8/group; * P

Techniques Used: Injection, Activation Assay

PPARγ antagonist, GW9662, reversed the suppressive effects of 12(S)-HETE and 15(S)-HETE on COX-2, iNOS, and NF-κB. Focal ischemia was induced by transient MCAO. Rats were injected ip with increasing concentrations (1, 2, and 4 mg/kg) of GW9662 30 min before the exposure to 12(S)- or 15(S)-HETE (20 μg) and MCAO. The COX-2 and iNOS protein levels, NO content, and NF-κB activation in ipsilateral (ischemic) cortex were estimated at 24 h post-MCAO. Data are expressed as mean ± SEM; n = 8/group; # P
Figure Legend Snippet: PPARγ antagonist, GW9662, reversed the suppressive effects of 12(S)-HETE and 15(S)-HETE on COX-2, iNOS, and NF-κB. Focal ischemia was induced by transient MCAO. Rats were injected ip with increasing concentrations (1, 2, and 4 mg/kg) of GW9662 30 min before the exposure to 12(S)- or 15(S)-HETE (20 μg) and MCAO. The COX-2 and iNOS protein levels, NO content, and NF-κB activation in ipsilateral (ischemic) cortex were estimated at 24 h post-MCAO. Data are expressed as mean ± SEM; n = 8/group; # P

Techniques Used: Injection, Activation Assay

13) Product Images from "Inhibition of TNF in the Brain Reverses Alterations in RAS Components and Attenuates Angiotensin II-Induced Hypertension"

Article Title: Inhibition of TNF in the Brain Reverses Alterations in RAS Components and Attenuates Angiotensin II-Induced Hypertension

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063847

Effect of central TNF blockade on iNOS and nNOS mRNA and protein expression in the PVN. Ang II-infused rats demonstrated an increase in the mRNA and protein expression of iNOS, and decrease in expression of nNOS within the PVN. These alterations were reversed by TNF blockade with ICV etanercept treatment. R.E. Relative expression, n = 8–12 per group, * p
Figure Legend Snippet: Effect of central TNF blockade on iNOS and nNOS mRNA and protein expression in the PVN. Ang II-infused rats demonstrated an increase in the mRNA and protein expression of iNOS, and decrease in expression of nNOS within the PVN. These alterations were reversed by TNF blockade with ICV etanercept treatment. R.E. Relative expression, n = 8–12 per group, * p

Techniques Used: Expressing

14) Product Images from "HemoHIM, a herbal preparation, alleviates airway inflammation caused by cigarette smoke and lipopolysaccharide"

Article Title: HemoHIM, a herbal preparation, alleviates airway inflammation caused by cigarette smoke and lipopolysaccharide

Journal: Laboratory Animal Research

doi: 10.5625/lar.2017.33.1.40

HemoHIM inhibited the iNOS and phosphorylation of ERK expression in lung tissue. (A) Expression of iNOS. (B) Phosphorylation of ERK. (C and D) Quantitative analysis of iNOS expression and phosphorylation of ERK expression. NC: Non-induced mice; CS+LPS: CS and LPS induced mice; ROF: roflumilast (10 mg/kg) and CS and LPS induced mice; H50: HemoHIM (50 mg/kg) and CS and LPS induced mice; H100 (100 mg/kg) and CS and LPS induced mice. The values are expressed as the means±SD. # Significantly different from the control mice, P
Figure Legend Snippet: HemoHIM inhibited the iNOS and phosphorylation of ERK expression in lung tissue. (A) Expression of iNOS. (B) Phosphorylation of ERK. (C and D) Quantitative analysis of iNOS expression and phosphorylation of ERK expression. NC: Non-induced mice; CS+LPS: CS and LPS induced mice; ROF: roflumilast (10 mg/kg) and CS and LPS induced mice; H50: HemoHIM (50 mg/kg) and CS and LPS induced mice; H100 (100 mg/kg) and CS and LPS induced mice. The values are expressed as the means±SD. # Significantly different from the control mice, P

Techniques Used: Expressing, Mouse Assay

15) Product Images from "Myeloid cell-derived inducible nitric oxide synthase suppresses M1 macrophage polarization"

Article Title: Myeloid cell-derived inducible nitric oxide synthase suppresses M1 macrophage polarization

Journal: Nature Communications

doi: 10.1038/ncomms7676

NO suppresses iNOS expression in macrophages. ( a ) BMDMs from WT and iNOS −/− mice were stimulated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) in the presence of SNAP (500 μM) or L-NIL (40 μM) overnight. The cell lysates were prepared and western blotting was performed for the analysis of iNOS protein expression. β-Actin expression was used as a control. Each bar represents mean±s.d. from three independent experiments, one-way ANOVA with a Bonferroni correction, * P
Figure Legend Snippet: NO suppresses iNOS expression in macrophages. ( a ) BMDMs from WT and iNOS −/− mice were stimulated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) in the presence of SNAP (500 μM) or L-NIL (40 μM) overnight. The cell lysates were prepared and western blotting was performed for the analysis of iNOS protein expression. β-Actin expression was used as a control. Each bar represents mean±s.d. from three independent experiments, one-way ANOVA with a Bonferroni correction, * P

Techniques Used: Expressing, Mouse Assay, Western Blot

NO induces nitration of tyrosine residues of IRF5 protein in macrophages. ( a ) RAW264.7 macrophages were pretreated with SNAP (500 μM) for 30 min and the cells were then activated with IFN-γ (10 ng ml −1 ) for various time intervals (10, 20, 30 and 60 min). Western blotting was performed for the analysis of STAT1 phosphorylation. β-Actin was used as a control. ( b ) RAW264.7 macrophages were pretreated with SNAP (500 μM) for 30 min, and the cells were then activated with IFN-γ (10 ng ml −1 ) for various time intervals (10, 20, 30 and 60 min). The cytosolic fraction and nuclear fraction of protein was prepared, and western blotting was performed for the analysis of phosphorylated STAT1 and STAT1 protein. ( c ) BMDMs from WT mice were stimulated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) in the presence of SNAP (500 μM) for 6 h, followed by ChIP assay. Three micrograms of an anti- phosphorylated STAT1 antibody or isotype-matched IgG as control antibody was used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the STAT1-binding site of the iNOS promoter region. Each bar represents mean±s.d. from three independent experiments, unpaired Student’s t -test, * P
Figure Legend Snippet: NO induces nitration of tyrosine residues of IRF5 protein in macrophages. ( a ) RAW264.7 macrophages were pretreated with SNAP (500 μM) for 30 min and the cells were then activated with IFN-γ (10 ng ml −1 ) for various time intervals (10, 20, 30 and 60 min). Western blotting was performed for the analysis of STAT1 phosphorylation. β-Actin was used as a control. ( b ) RAW264.7 macrophages were pretreated with SNAP (500 μM) for 30 min, and the cells were then activated with IFN-γ (10 ng ml −1 ) for various time intervals (10, 20, 30 and 60 min). The cytosolic fraction and nuclear fraction of protein was prepared, and western blotting was performed for the analysis of phosphorylated STAT1 and STAT1 protein. ( c ) BMDMs from WT mice were stimulated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) in the presence of SNAP (500 μM) for 6 h, followed by ChIP assay. Three micrograms of an anti- phosphorylated STAT1 antibody or isotype-matched IgG as control antibody was used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the STAT1-binding site of the iNOS promoter region. Each bar represents mean±s.d. from three independent experiments, unpaired Student’s t -test, * P

Techniques Used: Nitration, Western Blot, Mouse Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Binding Assay

NO suppresses IRF5 DNA binding activity. ( a ) BMDMs from WT and iNOS −/− mice were stimulated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) in the presence of SNAP (500 μM) or L-NIL (40 μM) overnight. The cell lysates were prepared and western blotting was performed for the analysis of protein expression of indicated genes. ( b ) BMDMs were activated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) in the presence of SNAP (500 μM) for 6 h and total cellular RNA was extracted. qPCR was performed for the analysis of mRNA expression of IRF5 and IRF4. ( c ) BMDMs were transfected with IRF5 siRNA or control siRNA, and the cells were then stimulated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) overnight. The cell lysates were prepared and western blotting was performed for the analysis of IRF5 protein expression. ( d ) BMDMs were transfected with IRF5 siRNA or control siRNA, and the cells were then stimulated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) overnight. The cells were stained for intracellular IL-12 and analysed by flow cytometry. Representative FACS dot plots gated on CD11b + cells, and the percentage of IL-12-producing CD11b + cells is shown. IL-12/23 p40 production was determined by ELISA. Each bar represents mean±s.d. from three independent experiments. ( e ) BMDMs from WT and iNOS −/− mice were stimulated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) in the presence of SNAP (500 μM) or L-NIL (40 μM) overnight. The cytosolic fraction and nuclear fraction of protein was prepared, and western blotting was performed for the analysis of IRF5 protein expression (upper panel). BMDMs from WT and iNOS −/− mice were stimulated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) in the presence of SNAP (500 μM) or L-NIL overnight, followed by ChIP assay. Three micrograms of an anti-IRF5 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the IRF5-binding site of the IL-12 promoter region (lower panel). Each bar represents mean±s.d. from three independent experiments, unpaired Student’s t -test, * P
Figure Legend Snippet: NO suppresses IRF5 DNA binding activity. ( a ) BMDMs from WT and iNOS −/− mice were stimulated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) in the presence of SNAP (500 μM) or L-NIL (40 μM) overnight. The cell lysates were prepared and western blotting was performed for the analysis of protein expression of indicated genes. ( b ) BMDMs were activated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) in the presence of SNAP (500 μM) for 6 h and total cellular RNA was extracted. qPCR was performed for the analysis of mRNA expression of IRF5 and IRF4. ( c ) BMDMs were transfected with IRF5 siRNA or control siRNA, and the cells were then stimulated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) overnight. The cell lysates were prepared and western blotting was performed for the analysis of IRF5 protein expression. ( d ) BMDMs were transfected with IRF5 siRNA or control siRNA, and the cells were then stimulated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) overnight. The cells were stained for intracellular IL-12 and analysed by flow cytometry. Representative FACS dot plots gated on CD11b + cells, and the percentage of IL-12-producing CD11b + cells is shown. IL-12/23 p40 production was determined by ELISA. Each bar represents mean±s.d. from three independent experiments. ( e ) BMDMs from WT and iNOS −/− mice were stimulated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) in the presence of SNAP (500 μM) or L-NIL (40 μM) overnight. The cytosolic fraction and nuclear fraction of protein was prepared, and western blotting was performed for the analysis of IRF5 protein expression (upper panel). BMDMs from WT and iNOS −/− mice were stimulated with IFN-γ (10 ng ml −1 ) and LPS (200 ng ml −1 ) in the presence of SNAP (500 μM) or L-NIL overnight, followed by ChIP assay. Three micrograms of an anti-IRF5 antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the IRF5-binding site of the IL-12 promoter region (lower panel). Each bar represents mean±s.d. from three independent experiments, unpaired Student’s t -test, * P

Techniques Used: Binding Assay, Activity Assay, Mouse Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Transfection, Staining, Flow Cytometry, Cytometry, FACS, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction

NO induces nitration of tyrosine residues of IRF5 protein in macrophages. ( a ) WT or iNOS −/− mice were injected (i.p.) with LPS (200 μg/mouse) for 6 or 12 h. Mice were then killed and spleen cells were prepared. The cells were stained for IRF5 and nitrotyrosine and analysed by flow cytometry. Representative FACS dot plots gated on CD11b + cells, and the percentages of IRF5 and nitrotyrosine-positive CD11b + cells are shown. Each bar represents mean±s.d. from three independent experiments, unpaired Student’s t -test, ** P
Figure Legend Snippet: NO induces nitration of tyrosine residues of IRF5 protein in macrophages. ( a ) WT or iNOS −/− mice were injected (i.p.) with LPS (200 μg/mouse) for 6 or 12 h. Mice were then killed and spleen cells were prepared. The cells were stained for IRF5 and nitrotyrosine and analysed by flow cytometry. Representative FACS dot plots gated on CD11b + cells, and the percentages of IRF5 and nitrotyrosine-positive CD11b + cells are shown. Each bar represents mean±s.d. from three independent experiments, unpaired Student’s t -test, ** P

Techniques Used: Nitration, Mouse Assay, Injection, Staining, Flow Cytometry, Cytometry, FACS

16) Product Images from "Isolation of Novel Sesquiterpeniods and Anti-neuroinflammatory Metabolites from Nardostachys jatamansi"

Article Title: Isolation of Novel Sesquiterpeniods and Anti-neuroinflammatory Metabolites from Nardostachys jatamansi

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

doi: 10.3390/molecules23092367

The effects of compounds ( A ) 3 , ( B ) 4 , and ( C ) 8 on the expression of iNOS and COX-2 proteins in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. (A–C) The cells were pretreated for 3 h with the indicated concentrations of compounds 3 , 4 , and 8, and then stimulated for 24 h with LPS (1.0 μg/mL). The data are presented as the means ± SD of three experiments. The band intensity was quantified by densitometry and normalized to the intensity of the β-actin band; lower panel, summarized bar graphs show band intensity presented as ratio of targeting protein over β-actin. The significance of the comparison against the LPS-treated group is indicated as follows: *** p
Figure Legend Snippet: The effects of compounds ( A ) 3 , ( B ) 4 , and ( C ) 8 on the expression of iNOS and COX-2 proteins in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. (A–C) The cells were pretreated for 3 h with the indicated concentrations of compounds 3 , 4 , and 8, and then stimulated for 24 h with LPS (1.0 μg/mL). The data are presented as the means ± SD of three experiments. The band intensity was quantified by densitometry and normalized to the intensity of the β-actin band; lower panel, summarized bar graphs show band intensity presented as ratio of targeting protein over β-actin. The significance of the comparison against the LPS-treated group is indicated as follows: *** p

Techniques Used: Expressing

17) Product Images from "S-nitrosylation of β-arrestins biases receptor signaling and confers ligand independence"

Article Title: S-nitrosylation of β-arrestins biases receptor signaling and confers ligand independence

Journal: Molecular cell

doi: 10.1016/j.molcel.2018.03.034

Dimerization of βarr1 and of βarr2 Induced by Expression of nNOS or iNOS, Dependent upon Cys 251/253
Figure Legend Snippet: Dimerization of βarr1 and of βarr2 Induced by Expression of nNOS or iNOS, Dependent upon Cys 251/253

Techniques Used: Expressing

S-nitrosylation by nNOS and by iNOS of Cys 251/253 within βarr1/2 and Association of n/iNOS with βarr1/2
Figure Legend Snippet: S-nitrosylation by nNOS and by iNOS of Cys 251/253 within βarr1/2 and Association of n/iNOS with βarr1/2

Techniques Used:

18) Product Images from "Pseudane-VII Isolated from Pseudoalteromonas sp. M2 Ameliorates LPS-Induced Inflammatory Response In Vitro and In Vivo"

Article Title: Pseudane-VII Isolated from Pseudoalteromonas sp. M2 Ameliorates LPS-Induced Inflammatory Response In Vitro and In Vivo

Journal: Marine Drugs

doi: 10.3390/md15110336

Pseudane-VII suppressed LPS-induced pro-inflammatory enzymes in RAW 264.7 macrophages. The inos and cox-2 mRNA and protein expression were determined by: RT-PCR ( A ); and Western blot analysis ( B ). RAW 264.7 cells were pre-treated with various concentrations of pseudane-VII (0–6 μM) for 2 h and stimulated with LPS (200 ng/mL) for 6 h (RT-PCR) or 24 h (Western blot). Pseudane-VII treatment reduced inos and cox-2 mRNA levels, as well as iNOS and COX-2 protein level. The respective internal controls were gapdh and β-actin. The data are representative of four separate experiments.
Figure Legend Snippet: Pseudane-VII suppressed LPS-induced pro-inflammatory enzymes in RAW 264.7 macrophages. The inos and cox-2 mRNA and protein expression were determined by: RT-PCR ( A ); and Western blot analysis ( B ). RAW 264.7 cells were pre-treated with various concentrations of pseudane-VII (0–6 μM) for 2 h and stimulated with LPS (200 ng/mL) for 6 h (RT-PCR) or 24 h (Western blot). Pseudane-VII treatment reduced inos and cox-2 mRNA levels, as well as iNOS and COX-2 protein level. The respective internal controls were gapdh and β-actin. The data are representative of four separate experiments.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

19) Product Images from "Malvidin Protects WI-38 Human Fibroblast Cells Against Stress-induced Premature Senescence"

Article Title: Malvidin Protects WI-38 Human Fibroblast Cells Against Stress-induced Premature Senescence

Journal: Journal of Cancer Prevention

doi: 10.15430/JCP.2016.21.1.32

Effect of malvidin on protein expression of inducible nitric oxide (iNOS), COX-2, and NF-κB in WI-38 cells under H 2 O 2 -induced premature senescence. Cells were incubated with malvidin for 48 hours and lysed. Cellular and nuclear proteins were separated by SDS PAGE and transferred onto nitrocellulose membranes, which were probed with anti-NF-κB, anti-COX-2, and anti-iNOS antibodies. (A) Proteins were visualized using an enhanced chemiluminescence detection system. Actin was used as an internal control. Graphs represent relative expression of (B) COX-2, (C) iNOS, and (D) nuclear NF-κB to actin expression. PSC, premature senescence control. a∼d Means with the different letters are significantly different ( P
Figure Legend Snippet: Effect of malvidin on protein expression of inducible nitric oxide (iNOS), COX-2, and NF-κB in WI-38 cells under H 2 O 2 -induced premature senescence. Cells were incubated with malvidin for 48 hours and lysed. Cellular and nuclear proteins were separated by SDS PAGE and transferred onto nitrocellulose membranes, which were probed with anti-NF-κB, anti-COX-2, and anti-iNOS antibodies. (A) Proteins were visualized using an enhanced chemiluminescence detection system. Actin was used as an internal control. Graphs represent relative expression of (B) COX-2, (C) iNOS, and (D) nuclear NF-κB to actin expression. PSC, premature senescence control. a∼d Means with the different letters are significantly different ( P

Techniques Used: Expressing, Incubation, SDS Page

20) Product Images from "Anti-inflammatory activity of edible oyster mushroom is mediated through the inhibition of NF-?B and AP-1 signaling"

Article Title: Anti-inflammatory activity of edible oyster mushroom is mediated through the inhibition of NF-?B and AP-1 signaling

Journal: Nutrition Journal

doi: 10.1186/1475-2891-10-52

Effect of OMC on LPS-induced PGE2 and NO secretion and COX-2 and iNOS expression in RAW264.7 cells . (A) PGE 2 and (C) NO secretion were determined in cell culture media from RAW264.7 cells treated with OMC and LPS as described in Materials and Methods . The data are means ± S.D. of two independent experiments, repeated minimally twice. Means without a common letter differ, P
Figure Legend Snippet: Effect of OMC on LPS-induced PGE2 and NO secretion and COX-2 and iNOS expression in RAW264.7 cells . (A) PGE 2 and (C) NO secretion were determined in cell culture media from RAW264.7 cells treated with OMC and LPS as described in Materials and Methods . The data are means ± S.D. of two independent experiments, repeated minimally twice. Means without a common letter differ, P

Techniques Used: Expressing, Cell Culture

21) Product Images from "IRAK-M Deficiency Exacerbates Ischemic Neurovascular Injuries in Experimental Stroke Mice"

Article Title: IRAK-M Deficiency Exacerbates Ischemic Neurovascular Injuries in Experimental Stroke Mice

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2018.00504

Effects of IRAK-M deletion on protein level of ischemia-induced inflammatory cytokines in mouse brains. (A–E) Representative Western blot images of COX-2 (A) , TNF-α (B) , IL-10 (C) , NLRP-3 (D) and iNOS (E) at 4 h and 24 h after stroke in KO and WT mice. β-Actin was used as the loading control. (F) Representative Western blot images of NF-κB P65 in nuclei in the ischemic hemisphere of KO and WT mice at 24 h after reperfusion. The relative protein level was quantified and normalized to β-actin using ImageJ. n = 5 per group. ∗ P
Figure Legend Snippet: Effects of IRAK-M deletion on protein level of ischemia-induced inflammatory cytokines in mouse brains. (A–E) Representative Western blot images of COX-2 (A) , TNF-α (B) , IL-10 (C) , NLRP-3 (D) and iNOS (E) at 4 h and 24 h after stroke in KO and WT mice. β-Actin was used as the loading control. (F) Representative Western blot images of NF-κB P65 in nuclei in the ischemic hemisphere of KO and WT mice at 24 h after reperfusion. The relative protein level was quantified and normalized to β-actin using ImageJ. n = 5 per group. ∗ P

Techniques Used: Western Blot, Mouse Assay

22) Product Images from "Curcumin Alleviates Matrix Metalloproteinase-3 and -9 Activities during Eradication of Helicobacter pylori Infection in Cultured Cells and Mice"

Article Title: Curcumin Alleviates Matrix Metalloproteinase-3 and -9 Activities during Eradication of Helicobacter pylori Infection in Cultured Cells and Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0016306

Regulation of MMP-3 and -9 via NF-kB dependent mechanism by curcumin-therapy in Hp-infected mice. Two weeks SS1-infected mice groups were treated with curcumin or TT or only-antibiotics for 7-days and the expressions of IL-1β, TNF-α, IL-8, and iNOS proteins were assessed by Western blot ( A ). Histographic representation of protein fold changes from the above blots and two other representative blots from independent experiments ( B ). Error bars = ±SEM. *, p
Figure Legend Snippet: Regulation of MMP-3 and -9 via NF-kB dependent mechanism by curcumin-therapy in Hp-infected mice. Two weeks SS1-infected mice groups were treated with curcumin or TT or only-antibiotics for 7-days and the expressions of IL-1β, TNF-α, IL-8, and iNOS proteins were assessed by Western blot ( A ). Histographic representation of protein fold changes from the above blots and two other representative blots from independent experiments ( B ). Error bars = ±SEM. *, p

Techniques Used: Infection, Mouse Assay, Western Blot

23) Product Images from "Extra-Esophageal Pepsin from Stomach Refluxate Promoted Tonsil Hypertrophy"

Article Title: Extra-Esophageal Pepsin from Stomach Refluxate Promoted Tonsil Hypertrophy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0152336

Localization of TGF- β1 and iNOS-positive cells in the tonsil sections. TGF- β1 and iNOS-positive cells were found in the crypt epithelium (A and B), surrounding germinal centers (C and D), and surrounding the lymphoid follicle with excessive developing fibrosis (E and F). Inserts in each image was magnified (A’-F’). Scale bar, 50 μm.
Figure Legend Snippet: Localization of TGF- β1 and iNOS-positive cells in the tonsil sections. TGF- β1 and iNOS-positive cells were found in the crypt epithelium (A and B), surrounding germinal centers (C and D), and surrounding the lymphoid follicle with excessive developing fibrosis (E and F). Inserts in each image was magnified (A’-F’). Scale bar, 50 μm.

Techniques Used:

24) Product Images from "Propionyl-L-Carnitine Enhances Wound Healing and Counteracts Microvascular Endothelial Cell Dysfunction"

Article Title: Propionyl-L-Carnitine Enhances Wound Healing and Counteracts Microvascular Endothelial Cell Dysfunction

Journal: PLoS ONE

doi: 10.1371/journal.pone.0140697

PLC ameliorates cell function in serum-deprived HMVECs. (A) Real-time PCR for PlGF mRNA in serum-deprived PBS-treated (vehicle) or PLC-treated HMVECs at different times. (B) PlGF protein concentration assessed by means of ELISA in treated cells. (C, D) Real-time PCR for Flt-1 and KDR transcripts. (E, F) Real-time PCR for eNOS and iNOS mRNA. (G) NO measurement in vehicle or PLC-treated cells at different times. (H, I) Real-time PCR for VCAM-1 and ICAM-1transcripts. (J) Leukocyte adhesion assay on vehicle and PLC-treated HMVECs and (K) representative microphotographs at 12h, magnification 100X. t-Student: * and ** indicate p
Figure Legend Snippet: PLC ameliorates cell function in serum-deprived HMVECs. (A) Real-time PCR for PlGF mRNA in serum-deprived PBS-treated (vehicle) or PLC-treated HMVECs at different times. (B) PlGF protein concentration assessed by means of ELISA in treated cells. (C, D) Real-time PCR for Flt-1 and KDR transcripts. (E, F) Real-time PCR for eNOS and iNOS mRNA. (G) NO measurement in vehicle or PLC-treated cells at different times. (H, I) Real-time PCR for VCAM-1 and ICAM-1transcripts. (J) Leukocyte adhesion assay on vehicle and PLC-treated HMVECs and (K) representative microphotographs at 12h, magnification 100X. t-Student: * and ** indicate p

Techniques Used: Planar Chromatography, Cell Function Assay, Real-time Polymerase Chain Reaction, Protein Concentration, Enzyme-linked Immunosorbent Assay, Cell Adhesion Assay

25) Product Images from "Macrophage polarization and activation at the interface of multi-walled carbon nanotube-induced pulmonary inflammation and fibrosis"

Article Title: Macrophage polarization and activation at the interface of multi-walled carbon nanotube-induced pulmonary inflammation and fibrosis

Journal: Nanotoxicology

doi: 10.1080/17435390.2018.1425501

Activation of M1 and M2 macrophages. Wild-type mice were exposed to DM or 40 μg MWCNTs for 1, 3, or 7 days. (A) Immunoblotting. iNOS was detected as a functional marker for M1 macrophage activation and ARG1 for M2 macrophage activation ( n = 2). (B) M1 activation examined by double immunofluorescence staining of iNOS (red) and F4/80 (green). (C) M2 activation examined by double immunofluorescence staining of ARG1 (red) and F4/80 (green). Blue indicates nuclear staining (scale bar: 20 μm). Representative double positive cells are marked with arrows. Quantification of double positive cells is shown as mean ± SD ( n = 4).
Figure Legend Snippet: Activation of M1 and M2 macrophages. Wild-type mice were exposed to DM or 40 μg MWCNTs for 1, 3, or 7 days. (A) Immunoblotting. iNOS was detected as a functional marker for M1 macrophage activation and ARG1 for M2 macrophage activation ( n = 2). (B) M1 activation examined by double immunofluorescence staining of iNOS (red) and F4/80 (green). (C) M2 activation examined by double immunofluorescence staining of ARG1 (red) and F4/80 (green). Blue indicates nuclear staining (scale bar: 20 μm). Representative double positive cells are marked with arrows. Quantification of double positive cells is shown as mean ± SD ( n = 4).

Techniques Used: Activation Assay, Mouse Assay, Functional Assay, Marker, Double Immunofluorescence Staining, Staining

26) Product Images from "Crosstalk between PKC? and the IL4/Stat6 pathway during T-cell-mediated hepatitis"

Article Title: Crosstalk between PKC? and the IL4/Stat6 pathway during T-cell-mediated hepatitis

Journal: The EMBO Journal

doi: 10.1038/sj.emboj.7600468

Impaired liver NF-κB signaling in ConA-injected PKCζ−/− mice. ( A ) Levels of iNOS were determined by immunoblot analysis of extracts from mice injected with 12 μg/g of ConA for 8 h. Results show representative ( n =5) autoradiographs of one mouse untreated and two mice injected with ConA either WT or PKCζ KO. ( B ) Nuclear extracts from the above experiment were analyzed by EMSA using a κB probe (left panel) or an Oct1 probe (right panel), as a negative control. ( C ) Liver IKK activity was also determined in extracts from the above experiment.
Figure Legend Snippet: Impaired liver NF-κB signaling in ConA-injected PKCζ−/− mice. ( A ) Levels of iNOS were determined by immunoblot analysis of extracts from mice injected with 12 μg/g of ConA for 8 h. Results show representative ( n =5) autoradiographs of one mouse untreated and two mice injected with ConA either WT or PKCζ KO. ( B ) Nuclear extracts from the above experiment were analyzed by EMSA using a κB probe (left panel) or an Oct1 probe (right panel), as a negative control. ( C ) Liver IKK activity was also determined in extracts from the above experiment.

Techniques Used: Injection, Mouse Assay, Negative Control, Activity Assay

27) Product Images from "Neuroprotective Effects of a Butanol Fraction of Rosa hybrida Petals in a Middle Cerebral Artery Occlusion Model"

Article Title: Neuroprotective Effects of a Butanol Fraction of Rosa hybrida Petals in a Middle Cerebral Artery Occlusion Model

Journal: Biomolecules & Therapeutics

doi: 10.4062/biomolther.2013.067

Effects of white rose petal extract-butanol fraction (WRPEBF) on the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and glial fibrillary acidic protein (GFAP) increased by middle cerebral artery occlusion (MCAO). The production of enzymes and GFAP proteins was analyzed by Western blotting (A), and quantified relative to the amount of β-actin (B). a Significantly different from sham control ( p
Figure Legend Snippet: Effects of white rose petal extract-butanol fraction (WRPEBF) on the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and glial fibrillary acidic protein (GFAP) increased by middle cerebral artery occlusion (MCAO). The production of enzymes and GFAP proteins was analyzed by Western blotting (A), and quantified relative to the amount of β-actin (B). a Significantly different from sham control ( p

Techniques Used: Expressing, Western Blot

28) Product Images from "In Vitro Model of Neuroinflammation: Efficacy of Cannabigerol, a Non-Psychoactive Cannabinoid"

Article Title: In Vitro Model of Neuroinflammation: Efficacy of Cannabigerol, a Non-Psychoactive Cannabinoid

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19071992

CBG pre-treatment was able to reduce the levels of oxidative stress markers nitrotyrosine, iNOS and SOD1 in NSC-34 cells treated with the medium of LPS-stimulated RAW 264.7 macrophages. The treatment with the medium of LPS-stimulated RAW 264.7 macrophages induced the expression of nitrotyrosine, iNOS and SOD1, but CBG pre-treatment reduced their levels. The immunocytochemical assays were repeated three times. **** p
Figure Legend Snippet: CBG pre-treatment was able to reduce the levels of oxidative stress markers nitrotyrosine, iNOS and SOD1 in NSC-34 cells treated with the medium of LPS-stimulated RAW 264.7 macrophages. The treatment with the medium of LPS-stimulated RAW 264.7 macrophages induced the expression of nitrotyrosine, iNOS and SOD1, but CBG pre-treatment reduced their levels. The immunocytochemical assays were repeated three times. **** p

Techniques Used: Expressing

29) Product Images from "PKN2 in colon cancer cells inhibits M2 phenotype polarization of tumor-associated macrophages via regulating DUSP6-Erk1/2 pathway"

Article Title: PKN2 in colon cancer cells inhibits M2 phenotype polarization of tumor-associated macrophages via regulating DUSP6-Erk1/2 pathway

Journal: Molecular Cancer

doi: 10.1186/s12943-017-0747-z

Overexpression of PKN2 is associated with better clinical outcome and high M1 content in human colon cancer. a The protein expression of PKN2, CD68, CD206, CD86, CD163 and iNOS in a human colon cancer tissue array was detected by immunohistochemistry staining. Representative photos are shown (100X and 400X). b Number of CD68 + , CD206 + , CD86 + , CD163 + and iNOS + cells per low field in tissues from colon cancer patients at different stages. c PKN2 expression in normal colon tissue, polyp, adenoma and metastatic adenocarcinoma was measured using IHC. PKN2 expression scores were shown in ( d ). *, P
Figure Legend Snippet: Overexpression of PKN2 is associated with better clinical outcome and high M1 content in human colon cancer. a The protein expression of PKN2, CD68, CD206, CD86, CD163 and iNOS in a human colon cancer tissue array was detected by immunohistochemistry staining. Representative photos are shown (100X and 400X). b Number of CD68 + , CD206 + , CD86 + , CD163 + and iNOS + cells per low field in tissues from colon cancer patients at different stages. c PKN2 expression in normal colon tissue, polyp, adenoma and metastatic adenocarcinoma was measured using IHC. PKN2 expression scores were shown in ( d ). *, P

Techniques Used: Over Expression, Expressing, Immunohistochemistry, Staining

30) Product Images from "Ameliorative effect of taurine-chloramine in azathioprine-induced testicular damage; a deeper insight into the mechanism of protection"

Article Title: Ameliorative effect of taurine-chloramine in azathioprine-induced testicular damage; a deeper insight into the mechanism of protection

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/s12906-018-2272-z

Western blotting analysis of testicular inducible nitric oxide synthase ( a ; iNOS) and p65 nuclear factor-kappa B ( b ; NF-ƘB p65) in Control, Taurine-chloramine (TAU-CL), Azathioprine (AZA) and TAU-CL/AZA groups. Data are presented as mean ± SEM, a Significant change from the control group at p
Figure Legend Snippet: Western blotting analysis of testicular inducible nitric oxide synthase ( a ; iNOS) and p65 nuclear factor-kappa B ( b ; NF-ƘB p65) in Control, Taurine-chloramine (TAU-CL), Azathioprine (AZA) and TAU-CL/AZA groups. Data are presented as mean ± SEM, a Significant change from the control group at p

Techniques Used: Western Blot

31) Product Images from "In Vitro Model of Neuroinflammation: Efficacy of Cannabigerol, a Non-Psychoactive Cannabinoid"

Article Title: In Vitro Model of Neuroinflammation: Efficacy of Cannabigerol, a Non-Psychoactive Cannabinoid

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19071992

CBG pre-treatment was able to reduce the levels of oxidative stress markers nitrotyrosine, iNOS and SOD1 in NSC-34 cells treated with the medium of LPS-stimulated RAW 264.7 macrophages. The treatment with the medium of LPS-stimulated RAW 264.7 macrophages induced the expression of nitrotyrosine, iNOS and SOD1, but CBG pre-treatment reduced their levels. The immunocytochemical assays were repeated three times. **** p
Figure Legend Snippet: CBG pre-treatment was able to reduce the levels of oxidative stress markers nitrotyrosine, iNOS and SOD1 in NSC-34 cells treated with the medium of LPS-stimulated RAW 264.7 macrophages. The treatment with the medium of LPS-stimulated RAW 264.7 macrophages induced the expression of nitrotyrosine, iNOS and SOD1, but CBG pre-treatment reduced their levels. The immunocytochemical assays were repeated three times. **** p

Techniques Used: Expressing

32) Product Images from "T cell-derived inducible nitric oxide synthase switches off TH17 cell differentiation"

Article Title: T cell-derived inducible nitric oxide synthase switches off TH17 cell differentiation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20122494

NO suppresses RORγt-mediated IL-17 transcription. (A) Naive CD4 + T cells from WT or iNOS −/− mice were differentiated under T H 17 polarizing conditions for 3 d. Cells were then restimulated with PMA/ionomycin for 5 h, stained for intracellular RORγt, and analyzed by flow cytometry. Representative FACS dot plots gated on CD4 + cells and the percentages of RORγt-positive CD4 + cells are shown. (B) The cells prepared in A were lysed and cell lysates were prepared. The expression of RORγt and other indicated proteins was analyzed by Western blotting. (C) 293T cells were transfected with T7-tagged RORγt plasmid for 40 h in the presence of SNAP (10, 50, 100, or 200 µM). Cell lysates were collected and RORγt protein expression was analyzed by Western blotting. Alternatively, 293T cells were transfected with T7-tagged RORγt plasmid for 40 h in the presence of 100 µM SNAP. Nuclear protein was extracted and RORγt protein expression was analyzed by Western blotting. (D) Naive CD4 + T cells from WT or iNOS −/− mice were differentiated under T H 17 polarizing conditions in the presence of 50 µM SNAP or 0.5 mM L-NIL for 3 d. Cells were then restimulated with PMA/ionomycin for 5 h, stained for intracellular RORγt and nitrotyrosine, and analyzed by flow cytometry. Representative FACS dot plots are gated on CD4 + cells. (E) Naive CD4 + T cells from WT mice were cultured under T H 17-polarizing conditions in the presence of 50 µM SNAP or 0.5 mM L-NIL for 60 h, followed by ChIP assay. 3 µg anti-RORγt antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the CNS2 and −250 to −50 regions of the IL-17 promoter. Each bar represents mean ± SD from three independent experiments.
Figure Legend Snippet: NO suppresses RORγt-mediated IL-17 transcription. (A) Naive CD4 + T cells from WT or iNOS −/− mice were differentiated under T H 17 polarizing conditions for 3 d. Cells were then restimulated with PMA/ionomycin for 5 h, stained for intracellular RORγt, and analyzed by flow cytometry. Representative FACS dot plots gated on CD4 + cells and the percentages of RORγt-positive CD4 + cells are shown. (B) The cells prepared in A were lysed and cell lysates were prepared. The expression of RORγt and other indicated proteins was analyzed by Western blotting. (C) 293T cells were transfected with T7-tagged RORγt plasmid for 40 h in the presence of SNAP (10, 50, 100, or 200 µM). Cell lysates were collected and RORγt protein expression was analyzed by Western blotting. Alternatively, 293T cells were transfected with T7-tagged RORγt plasmid for 40 h in the presence of 100 µM SNAP. Nuclear protein was extracted and RORγt protein expression was analyzed by Western blotting. (D) Naive CD4 + T cells from WT or iNOS −/− mice were differentiated under T H 17 polarizing conditions in the presence of 50 µM SNAP or 0.5 mM L-NIL for 3 d. Cells were then restimulated with PMA/ionomycin for 5 h, stained for intracellular RORγt and nitrotyrosine, and analyzed by flow cytometry. Representative FACS dot plots are gated on CD4 + cells. (E) Naive CD4 + T cells from WT mice were cultured under T H 17-polarizing conditions in the presence of 50 µM SNAP or 0.5 mM L-NIL for 60 h, followed by ChIP assay. 3 µg anti-RORγt antibody or isotype-matched IgG as control antibody were used in the immunoprecipitation step. PCR was used to quantify the amount of precipitated DNA with primers flanking the CNS2 and −250 to −50 regions of the IL-17 promoter. Each bar represents mean ± SD from three independent experiments.

Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry, FACS, Expressing, Western Blot, Transfection, Plasmid Preparation, Cell Culture, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction

33) Product Images from "Cell permeable HMGB1-binding heptamer peptide ameliorates neurovascular complications associated with thrombolytic therapy in rats with transient ischemic stroke"

Article Title: Cell permeable HMGB1-binding heptamer peptide ameliorates neurovascular complications associated with thrombolytic therapy in rats with transient ischemic stroke

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-018-1267-5

HBHP alleviated inflammatory reactions in tPA-treated stroke rats. a ELISA data of serum IL-1β levels in stroke rats at 8 h after ischemia, n = 5–7. b Quantitative real-time PCR analysis of mRNA expressions of IL-1β, IL-6, IL-12b, and TNF-α in the peri-infarct cortex from 4.5 h MCAO rats or in the same regions of sham-operated rats, n = 3–4. c Western blot and quantified data of iNOS, COX-2, and IL-1β in ischemic ipsilateral hemispheres at 8 h after ischemia, n = 3–4. NS: not significant; * P
Figure Legend Snippet: HBHP alleviated inflammatory reactions in tPA-treated stroke rats. a ELISA data of serum IL-1β levels in stroke rats at 8 h after ischemia, n = 5–7. b Quantitative real-time PCR analysis of mRNA expressions of IL-1β, IL-6, IL-12b, and TNF-α in the peri-infarct cortex from 4.5 h MCAO rats or in the same regions of sham-operated rats, n = 3–4. c Western blot and quantified data of iNOS, COX-2, and IL-1β in ischemic ipsilateral hemispheres at 8 h after ischemia, n = 3–4. NS: not significant; * P

Techniques Used: Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot

34) Product Images from "Anti-inflammatory and neuroprotective effects of an orally active apocynin derivative in pre-clinical models of Parkinson's disease"

Article Title: Anti-inflammatory and neuroprotective effects of an orally active apocynin derivative in pre-clinical models of Parkinson's disease

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-9-241

Diapocynin attenuates inducible nitric oxide synthase (iNOS) expression in the substantia nigra (SN) of MPTP-treated mice. ( A ) Representative Western blots illustrating the expression of iNOS in SN. ( B ) Bar graph showing means of Western blot iNOS/β-actin ratios ± SEM in SN of 6 mice per group. ( C ) Double labeling of glial fibrillary acidic protein (GFAP) and iNOS, and ( D ) Iba-1 and iNOS in the SN region of ventral midbrain. Images were captured at 30× and 60× magnification. *** P
Figure Legend Snippet: Diapocynin attenuates inducible nitric oxide synthase (iNOS) expression in the substantia nigra (SN) of MPTP-treated mice. ( A ) Representative Western blots illustrating the expression of iNOS in SN. ( B ) Bar graph showing means of Western blot iNOS/β-actin ratios ± SEM in SN of 6 mice per group. ( C ) Double labeling of glial fibrillary acidic protein (GFAP) and iNOS, and ( D ) Iba-1 and iNOS in the SN region of ventral midbrain. Images were captured at 30× and 60× magnification. *** P

Techniques Used: Expressing, Mouse Assay, Western Blot, Labeling

35) Product Images from "The protective effects of Polygonum multiflorum stilbeneglycoside preconditioning in an ischemia/reperfusion model of HUVECs"

Article Title: The protective effects of Polygonum multiflorum stilbeneglycoside preconditioning in an ischemia/reperfusion model of HUVECs

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2010.7

Effect of PMS on the expression of iNOS and eNOS in HUVECs. HUVECs were incubated for 3 h with or without various concentrations of PMS (0.6×10 −11 , 1.2×10 −11 , 2.4×10 −11 mol/L). The expression of iNOS and eNOS were assessed by Western blotting as described in the Materials and methods section. (A) A representative Western blot for iNOS and eNOS; (B) Relative levels of iNOS protein as assessed by densitometry; (C) Relative levels of eNOS protein as assessed by densitometry. Quantitations were normalized to value obtained for β-actin protein expression. All data are presented as mean±SEM. n =8. a P > 0.05, c P
Figure Legend Snippet: Effect of PMS on the expression of iNOS and eNOS in HUVECs. HUVECs were incubated for 3 h with or without various concentrations of PMS (0.6×10 −11 , 1.2×10 −11 , 2.4×10 −11 mol/L). The expression of iNOS and eNOS were assessed by Western blotting as described in the Materials and methods section. (A) A representative Western blot for iNOS and eNOS; (B) Relative levels of iNOS protein as assessed by densitometry; (C) Relative levels of eNOS protein as assessed by densitometry. Quantitations were normalized to value obtained for β-actin protein expression. All data are presented as mean±SEM. n =8. a P > 0.05, c P

Techniques Used: Expressing, Incubation, Western Blot

36) Product Images from "Biliverdin Reductase A (BVRA) Mediates Macrophage Expression of Interleukin-10 in Injured Kidney"

Article Title: Biliverdin Reductase A (BVRA) Mediates Macrophage Expression of Interleukin-10 in Injured Kidney

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms160922621

BVRA over-expressed macrophages showed increased INOS and ARG-1 expression levels. INOS ( A ) and ARG-1 ( B ) mRNA expression were detected by qRT-PCR in over-expressed BVRA macrophages; n = 4, *** p
Figure Legend Snippet: BVRA over-expressed macrophages showed increased INOS and ARG-1 expression levels. INOS ( A ) and ARG-1 ( B ) mRNA expression were detected by qRT-PCR in over-expressed BVRA macrophages; n = 4, *** p

Techniques Used: Expressing, Quantitative RT-PCR

Macrophage polarization was induced successfully in vitro . Macrophages’ morphology was observed by light microscope, (M0) control macrophages (untreated macrophages); (M1) macrophages stimulated with GM-CSF; (M2) macrophages stimulated with M-CSF ( A ). Original magnification ×400. INOS ( B ), ARG-1( C ), IL-10 and TNF-α ( D ) mRNA expression were detected by qRT-PCR. IL-10 and TNF-α ( E ) protein expression were detected by ELISA. n = 4, ** p
Figure Legend Snippet: Macrophage polarization was induced successfully in vitro . Macrophages’ morphology was observed by light microscope, (M0) control macrophages (untreated macrophages); (M1) macrophages stimulated with GM-CSF; (M2) macrophages stimulated with M-CSF ( A ). Original magnification ×400. INOS ( B ), ARG-1( C ), IL-10 and TNF-α ( D ) mRNA expression were detected by qRT-PCR. IL-10 and TNF-α ( E ) protein expression were detected by ELISA. n = 4, ** p

Techniques Used: In Vitro, Light Microscopy, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

37) Product Images from "A Novel Role of Lactosylceramide in the Regulation of Lipopolysaccharide/Interferon-γ-Mediated Inducible Nitric Oxide Synthase Gene Expression: Implications for Neuroinflammatory Diseases"

Article Title: A Novel Role of Lactosylceramide in the Regulation of Lipopolysaccharide/Interferon-γ-Mediated Inducible Nitric Oxide Synthase Gene Expression: Implications for Neuroinflammatory Diseases

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1271-04.2004

The effect of LPS/IFN-γ stimulation on the biosynthesis of LacCer. A , Primary astrocytes were treated with [ 14 C]galactose overnight. After pretreatment with PDMP 0.5 hr before LPS/IFN-γ stimulation, the cells were harvested at the time points indicated, and LacCer was analyzed by HPTLC as described in Materials and Methods. B , The enzyme activity of LacCer synthase (GalT-2) was assayed as described in Materials and Methods, using cell lysates derived from cells stimulated with LPS/IFN-γ for various durations as shown. C , For the silencing of GalT-2 gene, the cells were transfected with either GalT-2 antisense DNA oligomer or its sequence-scrambled DNA oligomer (Scr) as described in Materials and Methods. At 48 hr after transfection GalT-2 protein levels were analyzed by immunoblot analysis, and [ 14 C]LacCer synthesis was examined in transfected and nontransfected cells. At 48 hr after transfection with AS oligonucleotides the cells were stimulated with LPS/IFN-γ and NO production ( D ), and the mRNA levels of iNOS, TNF-α, and IL-1β ( E ) were measured as described previously. Data are represented as the mean ± SD of three independent experiments. *** p
Figure Legend Snippet: The effect of LPS/IFN-γ stimulation on the biosynthesis of LacCer. A , Primary astrocytes were treated with [ 14 C]galactose overnight. After pretreatment with PDMP 0.5 hr before LPS/IFN-γ stimulation, the cells were harvested at the time points indicated, and LacCer was analyzed by HPTLC as described in Materials and Methods. B , The enzyme activity of LacCer synthase (GalT-2) was assayed as described in Materials and Methods, using cell lysates derived from cells stimulated with LPS/IFN-γ for various durations as shown. C , For the silencing of GalT-2 gene, the cells were transfected with either GalT-2 antisense DNA oligomer or its sequence-scrambled DNA oligomer (Scr) as described in Materials and Methods. At 48 hr after transfection GalT-2 protein levels were analyzed by immunoblot analysis, and [ 14 C]LacCer synthesis was examined in transfected and nontransfected cells. At 48 hr after transfection with AS oligonucleotides the cells were stimulated with LPS/IFN-γ and NO production ( D ), and the mRNA levels of iNOS, TNF-α, and IL-1β ( E ) were measured as described previously. Data are represented as the mean ± SD of three independent experiments. *** p

Techniques Used: High Performance Thin Layer Chromatography, Activity Assay, Derivative Assay, Transfection, Sequencing

38) Product Images from "Thymoquinone Defeats Diabetes-Induced Testicular Damage in Rats Targeting Antioxidant, Inflammatory and Aromatase Expression"

Article Title: Thymoquinone Defeats Diabetes-Induced Testicular Damage in Rats Targeting Antioxidant, Inflammatory and Aromatase Expression

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18050919

Western blotting analysis of iNOS ( A ), NF-κB-p65 ( B ), and Aromatase ( C ) in Control, Diabetic, Diabetic + TQ, and TQ groups. * p
Figure Legend Snippet: Western blotting analysis of iNOS ( A ), NF-κB-p65 ( B ), and Aromatase ( C ) in Control, Diabetic, Diabetic + TQ, and TQ groups. * p

Techniques Used: Western Blot

39) Product Images from "Macrophage polarization and activation at the interface of multi-walled carbon nanotube-induced pulmonary inflammation and fibrosis"

Article Title: Macrophage polarization and activation at the interface of multi-walled carbon nanotube-induced pulmonary inflammation and fibrosis

Journal: Nanotoxicology

doi: 10.1080/17435390.2018.1425501

Activation of M1 and M2 macrophages. Wild-type mice were exposed to DM or 40 μg MWCNTs for 1, 3, or 7 days. (A) Immunoblotting. iNOS was detected as a functional marker for M1 macrophage activation and ARG1 for M2 macrophage activation ( n = 2). (B) M1 activation examined by double immunofluorescence staining of iNOS (red) and F4/80 (green). (C) M2 activation examined by double immunofluorescence staining of ARG1 (red) and F4/80 (green). Blue indicates nuclear staining (scale bar: 20 μm). Representative double positive cells are marked with arrows. Quantification of double positive cells is shown as mean ± SD ( n = 4).
Figure Legend Snippet: Activation of M1 and M2 macrophages. Wild-type mice were exposed to DM or 40 μg MWCNTs for 1, 3, or 7 days. (A) Immunoblotting. iNOS was detected as a functional marker for M1 macrophage activation and ARG1 for M2 macrophage activation ( n = 2). (B) M1 activation examined by double immunofluorescence staining of iNOS (red) and F4/80 (green). (C) M2 activation examined by double immunofluorescence staining of ARG1 (red) and F4/80 (green). Blue indicates nuclear staining (scale bar: 20 μm). Representative double positive cells are marked with arrows. Quantification of double positive cells is shown as mean ± SD ( n = 4).

Techniques Used: Activation Assay, Mouse Assay, Functional Assay, Marker, Double Immunofluorescence Staining, Staining

40) Product Images from "Quercitrin from Toona sinensis (Juss.) M.Roem. Attenuates Acetaminophen-Induced Acute Liver Toxicity in HepG2 Cells and Mice through Induction of Antioxidant Machinery and Inhibition of Inflammation"

Article Title: Quercitrin from Toona sinensis (Juss.) M.Roem. Attenuates Acetaminophen-Induced Acute Liver Toxicity in HepG2 Cells and Mice through Induction of Antioxidant Machinery and Inhibition of Inflammation

Journal: Nutrients

doi: 10.3390/nu8070431

Effects of quercitrin on inflammatory response in APAP intoxicated mice. The hepatic inducible nitric oxide (iNOS) ( A ); cyclooxygenase 2 (COX-2) ( B ) and interleukin 1β (IL-1β) ( C ) levels were evaluated by Western blot analysis. Protein expression levels were normalized with β-actin. All data represent means ± SD of five mice in each group. Significant differences were ( # ) p
Figure Legend Snippet: Effects of quercitrin on inflammatory response in APAP intoxicated mice. The hepatic inducible nitric oxide (iNOS) ( A ); cyclooxygenase 2 (COX-2) ( B ) and interleukin 1β (IL-1β) ( C ) levels were evaluated by Western blot analysis. Protein expression levels were normalized with β-actin. All data represent means ± SD of five mice in each group. Significant differences were ( # ) p

Techniques Used: Mouse Assay, Western Blot, Expressing

41) Product Images from "Anti-neuroinflammatory effects of cudraflavanone A isolated from the chloroform fraction of Cudrania tricuspidata root bark"

Article Title: Anti-neuroinflammatory effects of cudraflavanone A isolated from the chloroform fraction of Cudrania tricuspidata root bark

Journal: Pharmaceutical Biology

doi: 10.1080/13880209.2018.1447972

Effects of cudraflavanone A on LPS-induced NO, PGE2 production, iNOS, COX-2 protein expression and COX-2 enzyme activity in BV2 cells. (A–C) Cells were pretreated with/without the indicated concentrations of cudraflavanone A for 3 h and then stimulated with LPS (1 µg/mL) for 24 h. Nitrite levels (A) were determined using the Griess reaction and PGE2 (B) was quantified by ELISA. * p
Figure Legend Snippet: Effects of cudraflavanone A on LPS-induced NO, PGE2 production, iNOS, COX-2 protein expression and COX-2 enzyme activity in BV2 cells. (A–C) Cells were pretreated with/without the indicated concentrations of cudraflavanone A for 3 h and then stimulated with LPS (1 µg/mL) for 24 h. Nitrite levels (A) were determined using the Griess reaction and PGE2 (B) was quantified by ELISA. * p

Techniques Used: Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay

42) Product Images from "Anti-inflammatory and neuroprotective effects of an orally active apocynin derivative in pre-clinical models of Parkinson's disease"

Article Title: Anti-inflammatory and neuroprotective effects of an orally active apocynin derivative in pre-clinical models of Parkinson's disease

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-9-241

Diapocynin attenuates inducible nitric oxide synthase (iNOS) expression in the substantia nigra (SN) of MPTP-treated mice. ( A ) Representative Western blots illustrating the expression of iNOS in SN. ( B ) Bar graph showing means of Western blot iNOS/β-actin ratios ± SEM in SN of 6 mice per group. ( C ) Double labeling of glial fibrillary acidic protein (GFAP) and iNOS, and ( D ) Iba-1 and iNOS in the SN region of ventral midbrain. Images were captured at 30× and 60× magnification. *** P
Figure Legend Snippet: Diapocynin attenuates inducible nitric oxide synthase (iNOS) expression in the substantia nigra (SN) of MPTP-treated mice. ( A ) Representative Western blots illustrating the expression of iNOS in SN. ( B ) Bar graph showing means of Western blot iNOS/β-actin ratios ± SEM in SN of 6 mice per group. ( C ) Double labeling of glial fibrillary acidic protein (GFAP) and iNOS, and ( D ) Iba-1 and iNOS in the SN region of ventral midbrain. Images were captured at 30× and 60× magnification. *** P

Techniques Used: Expressing, Mouse Assay, Western Blot, Labeling

43) Product Images from "Tongxinluo Inhibits Cyclooxygenase-2, Inducible Nitric Oxide Synthase, Hypoxia-inducible Factor-2α/Vascular Endothelial Growth Factor to Antagonize Injury in Hypoxia-stimulated Cardiac Microvascular Endothelial Cells"

Article Title: Tongxinluo Inhibits Cyclooxygenase-2, Inducible Nitric Oxide Synthase, Hypoxia-inducible Factor-2α/Vascular Endothelial Growth Factor to Antagonize Injury in Hypoxia-stimulated Cardiac Microvascular Endothelial Cells

Journal: Chinese Medical Journal

doi: 10.4103/0366-6999.155119

Effects of tongxinluo (TXL) on the hypoxia-induced protein content changes. (a) The protein contents of cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS), inducible nitric oxide synthase (iNOS), endothelial NOS, hypoxia-inducible factor (HIF)-1α, HIF-2α, and vascular endothelial growth factor (VEGF) were detected by Western blotting; (b) TXL decreased the hypoxia-induced COX-2, with increase of PGIS; (c) TXL decreased the hypoxia-induced iNOS; (d) TXL decreased the hypoxia-induced HIF-1α, HIF-2α, and VEGF. * P
Figure Legend Snippet: Effects of tongxinluo (TXL) on the hypoxia-induced protein content changes. (a) The protein contents of cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS), inducible nitric oxide synthase (iNOS), endothelial NOS, hypoxia-inducible factor (HIF)-1α, HIF-2α, and vascular endothelial growth factor (VEGF) were detected by Western blotting; (b) TXL decreased the hypoxia-induced COX-2, with increase of PGIS; (c) TXL decreased the hypoxia-induced iNOS; (d) TXL decreased the hypoxia-induced HIF-1α, HIF-2α, and VEGF. * P

Techniques Used: Western Blot

44) Product Images from "Levosimendan Inhibits Peroxidation in Hepatocytes by Modulating Apoptosis/Autophagy Interplay"

Article Title: Levosimendan Inhibits Peroxidation in Hepatocytes by Modulating Apoptosis/Autophagy Interplay

Journal: PLoS ONE

doi: 10.1371/journal.pone.0124742

Effects of levosimendan on eNOS (A) and iNOS activation (B) in hepatocytes subjected to peroxidation. In A and B, densitometric analysis of p-eNOS and iNOS and an example of lanes taken in one of 5 different experiments performed for each experimental protocol. eNOS = endothelial nitric oxide isoform; iNOS = inducible nitric oxide isoform; L = levosimendan; NM = L-NAME. The other abbreviations are as in Figs 1 – 4 . In A, b, d, e, f, g P
Figure Legend Snippet: Effects of levosimendan on eNOS (A) and iNOS activation (B) in hepatocytes subjected to peroxidation. In A and B, densitometric analysis of p-eNOS and iNOS and an example of lanes taken in one of 5 different experiments performed for each experimental protocol. eNOS = endothelial nitric oxide isoform; iNOS = inducible nitric oxide isoform; L = levosimendan; NM = L-NAME. The other abbreviations are as in Figs 1 – 4 . In A, b, d, e, f, g P

Techniques Used: Activation Assay

45) Product Images from "Nitro-Oxidative Stress after Neuronal Ischemia Induces Protein Nitrotyrosination and Cell Death"

Article Title: Nitro-Oxidative Stress after Neuronal Ischemia Induces Protein Nitrotyrosination and Cell Death

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2013/826143

Differential expression of mRNA NOS types occurs in different cell types. Cells were challenged with ischemia, and iNOS , eNOS, and nNOS mRNA expression were studied immediately at time 0, 12 and 24 h after the ischemic challenge. The expression of mRNA was assessed by semiquantitative RT-PCR, and bands were quantified by HPRT densitometric analysis in all cell types. Data are mean ± SEM values of 3 experiments in all cell types. * P
Figure Legend Snippet: Differential expression of mRNA NOS types occurs in different cell types. Cells were challenged with ischemia, and iNOS , eNOS, and nNOS mRNA expression were studied immediately at time 0, 12 and 24 h after the ischemic challenge. The expression of mRNA was assessed by semiquantitative RT-PCR, and bands were quantified by HPRT densitometric analysis in all cell types. Data are mean ± SEM values of 3 experiments in all cell types. * P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

NO is produced in different cell types by the different NOS types. Cells were challenged with ischemia and iNOS, eNOS, and nNOS protein levels were studied immediately at time 0, 12, and 24 h after the ischemic challenge. Densitometric analysis of the bands quantified NOS expression relative to tubulin in all cell types. Data are mean ± SEM values of 6 experiments for microglia and 3 experiments for neurons, endothelial cells, and myocytes. * P
Figure Legend Snippet: NO is produced in different cell types by the different NOS types. Cells were challenged with ischemia and iNOS, eNOS, and nNOS protein levels were studied immediately at time 0, 12, and 24 h after the ischemic challenge. Densitometric analysis of the bands quantified NOS expression relative to tubulin in all cell types. Data are mean ± SEM values of 6 experiments for microglia and 3 experiments for neurons, endothelial cells, and myocytes. * P

Techniques Used: Produced, Expressing

46) Product Images from "Anti-Inflammatory and Antinociceptive Effects of Ethyl Acetate Fraction of an Edible Red Macroalgae Sarcodia ceylanica"

Article Title: Anti-Inflammatory and Antinociceptive Effects of Ethyl Acetate Fraction of an Edible Red Macroalgae Sarcodia ceylanica

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18112437

Effects of PD1 on the carrageenan-induced upregulation of immunoreactivity of MPO (green), IL-1β (red) and iNOS (red) in rat paw tissue. Control ( A , D , G ), carrageenan ( B , E , H ), and carrageenan + PD1 ( C , F , I ) groups. Immunoreactivity quantitative results of MPO ( J ), IL-1β ( K ) and iNOS ( L ). In the quantitative figures ( J , K , L ), the horizontal axis shows the treatment groups, and the vertical axis shows the levels of immunoreactivity of the protein. Data are mean ± SEM with 6 rats per group; * p
Figure Legend Snippet: Effects of PD1 on the carrageenan-induced upregulation of immunoreactivity of MPO (green), IL-1β (red) and iNOS (red) in rat paw tissue. Control ( A , D , G ), carrageenan ( B , E , H ), and carrageenan + PD1 ( C , F , I ) groups. Immunoreactivity quantitative results of MPO ( J ), IL-1β ( K ) and iNOS ( L ). In the quantitative figures ( J , K , L ), the horizontal axis shows the treatment groups, and the vertical axis shows the levels of immunoreactivity of the protein. Data are mean ± SEM with 6 rats per group; * p

Techniques Used:

Effects of PD1 on expression of carrageenan-induced IL-1β (red) and iNOS (red) in leukocytes (MPO-positive cells; green) in rat paw tissues. The sections are show for control ( A , D ); carrageenan ( B , E ) and the carrageenan + PD1 ( C , F ). Double immunofluorescent staining revealed that PD1 significantly inhibited carrageenan-induced co-localization (arrows) of MPO with iNOS and IL-1β. Scale bar: 50 μm.
Figure Legend Snippet: Effects of PD1 on expression of carrageenan-induced IL-1β (red) and iNOS (red) in leukocytes (MPO-positive cells; green) in rat paw tissues. The sections are show for control ( A , D ); carrageenan ( B , E ) and the carrageenan + PD1 ( C , F ). Double immunofluorescent staining revealed that PD1 significantly inhibited carrageenan-induced co-localization (arrows) of MPO with iNOS and IL-1β. Scale bar: 50 μm.

Techniques Used: Expressing, Staining

47) Product Images from "Involvement of Heme Oxygenase-1 Induction in the Cytoprotective and Immunomodulatory Activities of Viola patrinii in Murine Hippocampal and Microglia Cells"

Article Title: Involvement of Heme Oxygenase-1 Induction in the Cytoprotective and Immunomodulatory Activities of Viola patrinii in Murine Hippocampal and Microglia Cells

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2012/128019

Effects of NNMBS275 on LPS-induced inducible iNOS and COX-2 protein expression and NO, PGE 2 , and proinflammatory cytokine levels in BV2 microglia. BV2 microglia were pretreated for 12 h with indicated concentrations of NNMBS275 and stimulated for 18 h with LPS (0.5 μ g/mL). Western blot analyses for iNOS and COX-2 expression (a and b) were performed as described in Section 2 . Representative blots of 3 independent experiments are shown. The concentrations of NO, PGE 2 , tumor necrosis TNF- α , and IL-1 β (c, d, e, and f) were determined as described under Section 2 . Data represent mean values of 3 experiments ± S.D. * P
Figure Legend Snippet: Effects of NNMBS275 on LPS-induced inducible iNOS and COX-2 protein expression and NO, PGE 2 , and proinflammatory cytokine levels in BV2 microglia. BV2 microglia were pretreated for 12 h with indicated concentrations of NNMBS275 and stimulated for 18 h with LPS (0.5 μ g/mL). Western blot analyses for iNOS and COX-2 expression (a and b) were performed as described in Section 2 . Representative blots of 3 independent experiments are shown. The concentrations of NO, PGE 2 , tumor necrosis TNF- α , and IL-1 β (c, d, e, and f) were determined as described under Section 2 . Data represent mean values of 3 experiments ± S.D. * P

Techniques Used: Expressing, Western Blot

48) Product Images from "Involvement of Heme Oxygenase-1 Induction in the Cytoprotective and Immunomodulatory Activities of Viola patrinii in Murine Hippocampal and Microglia Cells"

Article Title: Involvement of Heme Oxygenase-1 Induction in the Cytoprotective and Immunomodulatory Activities of Viola patrinii in Murine Hippocampal and Microglia Cells

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2012/128019

Effects of NNMBS275 on LPS-induced inducible iNOS and COX-2 protein expression and NO, PGE 2 , and proinflammatory cytokine levels in BV2 microglia. BV2 microglia were pretreated for 12 h with indicated concentrations of NNMBS275 and stimulated for 18 h with LPS (0.5 μ g/mL). Western blot analyses for iNOS and COX-2 expression (a and b) were performed as described in Section 2 . Representative blots of 3 independent experiments are shown. The concentrations of NO, PGE 2 , tumor necrosis TNF- α , and IL-1 β (c, d, e, and f) were determined as described under Section 2 . Data represent mean values of 3 experiments ± S.D. * P
Figure Legend Snippet: Effects of NNMBS275 on LPS-induced inducible iNOS and COX-2 protein expression and NO, PGE 2 , and proinflammatory cytokine levels in BV2 microglia. BV2 microglia were pretreated for 12 h with indicated concentrations of NNMBS275 and stimulated for 18 h with LPS (0.5 μ g/mL). Western blot analyses for iNOS and COX-2 expression (a and b) were performed as described in Section 2 . Representative blots of 3 independent experiments are shown. The concentrations of NO, PGE 2 , tumor necrosis TNF- α , and IL-1 β (c, d, e, and f) were determined as described under Section 2 . Data represent mean values of 3 experiments ± S.D. * P

Techniques Used: Expressing, Western Blot

49) Product Images from "Neuroprotective Effect of Artesunate in Experimental Model of Traumatic Brain Injury"

Article Title: Neuroprotective Effect of Artesunate in Experimental Model of Traumatic Brain Injury

Journal: Frontiers in Neurology

doi: 10.3389/fneur.2018.00590

Protective effect of artesunate treatment on cytokines and iNOS expression. (B,F) Showed a significant increase of expression for IL-1β and TNF-α respectively, 24 h after TBI injury compared with sham group (A,E) . When compared with vehicle group, brain section from mice treated with artesunate show a significant reduction in IL-1β and TNF-α respectively (C,G) , see densitometric analysis (D) for IL-1β, and (H) for TNF-α. The immunohistochemical analysis for iNOS, show that 24 h after TBI injury in the vehicle group there is a significant increase in iNOS expression as show in (J) compared with sham group (I) . While as show in (K) treatment with artesunate significantly reduce the iNOS expression compared to vehicle group, see densitometric analysis (L) . Data are expressed as Mean ± SEM from N = 10 Mice for each group. *** P
Figure Legend Snippet: Protective effect of artesunate treatment on cytokines and iNOS expression. (B,F) Showed a significant increase of expression for IL-1β and TNF-α respectively, 24 h after TBI injury compared with sham group (A,E) . When compared with vehicle group, brain section from mice treated with artesunate show a significant reduction in IL-1β and TNF-α respectively (C,G) , see densitometric analysis (D) for IL-1β, and (H) for TNF-α. The immunohistochemical analysis for iNOS, show that 24 h after TBI injury in the vehicle group there is a significant increase in iNOS expression as show in (J) compared with sham group (I) . While as show in (K) treatment with artesunate significantly reduce the iNOS expression compared to vehicle group, see densitometric analysis (L) . Data are expressed as Mean ± SEM from N = 10 Mice for each group. *** P

Techniques Used: Expressing, Mouse Assay, Immunohistochemistry

50) 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 the PGE 2 production ( A ) and protein expression of iNOS and COX-2 ( B ) in BV2 microglia stimulated with LPS. Cells were pretreated for 3 h with the indicated concentrations of cudratricusxanthone A ( 1 ) and then stimulated for 24 h with LPS (1 μg/mL). The LPS treatment was performed in the presence of compound. The concentrations of iNOS and COX-2 ( B ) were determined as described in the Experimental Section. Western blot analyses were performed as described in the Experimental Section, and representative blots of three independent experiments are shown. The band intensity was quantified by densitometry and normalized to β-actin, 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 the PGE 2 production ( A ) and protein expression of iNOS and COX-2 ( B ) in BV2 microglia stimulated with LPS. Cells were pretreated for 3 h with the indicated concentrations of cudratricusxanthone A ( 1 ) and then stimulated for 24 h with LPS (1 μg/mL). The LPS treatment was performed in the presence of compound. The concentrations of iNOS and COX-2 ( B ) were determined as described in the Experimental Section. Western blot analyses were performed as described in the Experimental Section, and representative blots of three independent experiments are shown. The band intensity was quantified by densitometry and normalized to β-actin, and the values are presented at the bottom of the each band. Relative data represent the means ± SDs of three experiments. * p

Techniques Used: Expressing, Western Blot

51) Product Images from "Deletion of lactate dehydrogenase-A in myeloid cells triggers antitumor immunity"

Article Title: Deletion of lactate dehydrogenase-A in myeloid cells triggers antitumor immunity

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-16-2938

Lack of LDH-A results in higher levels of M1 Mø markers A–C . Mø were isolated from Cre tm -LDH-A flfl (ko) and LDH-A flfl (wt) mice injected with tamoxifen (10 mg/ml in corn oil; every day for 4 days; i.p.). Differentiated Mø were polarized with LPS (100 ng/ml) and IFNγ (20 ng/ml) (M1) or IL-4 (20 ng/ml) (M2) for 3–5 days, Western blotting with antibody against LDH-A is shown in A . The number of CD197 (CCR7)-PE + /CD86-APC + (M1) or MMR-PE + (M2) cells were measured by flow cytometry ( B C ). Data are representative for 3 independent experiments in triplicates. D–E . Flow cytometry with the antibodies against M1/M2-like skewing in Mø polarized in the presence of DMSO (vehicle) or LDH-A inhibitor (10 µM) for 3 days. n=3. F–G . Representative immunofluorescence staining of K-Ras lungs with antibody against iNOS and F4.80 are shown in F. Quantification is shown in G. Lungs were harvested after 10 days after induction of Cre with tamoxifen in K-Ras mice treated with doxycycline diet for 13 weeks to induce lung carcinogenesis prior tamoxifen. n=3–4 mice per group. *p
Figure Legend Snippet: Lack of LDH-A results in higher levels of M1 Mø markers A–C . Mø were isolated from Cre tm -LDH-A flfl (ko) and LDH-A flfl (wt) mice injected with tamoxifen (10 mg/ml in corn oil; every day for 4 days; i.p.). Differentiated Mø were polarized with LPS (100 ng/ml) and IFNγ (20 ng/ml) (M1) or IL-4 (20 ng/ml) (M2) for 3–5 days, Western blotting with antibody against LDH-A is shown in A . The number of CD197 (CCR7)-PE + /CD86-APC + (M1) or MMR-PE + (M2) cells were measured by flow cytometry ( B C ). Data are representative for 3 independent experiments in triplicates. D–E . Flow cytometry with the antibodies against M1/M2-like skewing in Mø polarized in the presence of DMSO (vehicle) or LDH-A inhibitor (10 µM) for 3 days. n=3. F–G . Representative immunofluorescence staining of K-Ras lungs with antibody against iNOS and F4.80 are shown in F. Quantification is shown in G. Lungs were harvested after 10 days after induction of Cre with tamoxifen in K-Ras mice treated with doxycycline diet for 13 weeks to induce lung carcinogenesis prior tamoxifen. n=3–4 mice per group. *p

Techniques Used: Isolation, Mouse Assay, Injection, Western Blot, Flow Cytometry, Cytometry, Immunofluorescence, Staining

52) Product Images from "Deletion of lactate dehydrogenase-A in myeloid cells triggers antitumor immunity"

Article Title: Deletion of lactate dehydrogenase-A in myeloid cells triggers antitumor immunity

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-16-2938

Lack of LDH-A results in higher levels of M1 Mø markers A–C . Mø were isolated from Cre tm -LDH-A flfl (ko) and LDH-A flfl (wt) mice injected with tamoxifen (10 mg/ml in corn oil; every day for 4 days; i.p.). Differentiated Mø were polarized with LPS (100 ng/ml) and IFNγ (20 ng/ml) (M1) or IL-4 (20 ng/ml) (M2) for 3–5 days, Western blotting with antibody against LDH-A is shown in A . The number of CD197 (CCR7)-PE + /CD86-APC + (M1) or MMR-PE + (M2) cells were measured by flow cytometry ( B C ). Data are representative for 3 independent experiments in triplicates. D–E . Flow cytometry with the antibodies against M1/M2-like skewing in Mø polarized in the presence of DMSO (vehicle) or LDH-A inhibitor (10 µM) for 3 days. n=3. F–G . Representative immunofluorescence staining of K-Ras lungs with antibody against iNOS and F4.80 are shown in F. Quantification is shown in G. Lungs were harvested after 10 days after induction of Cre with tamoxifen in K-Ras mice treated with doxycycline diet for 13 weeks to induce lung carcinogenesis prior tamoxifen. n=3–4 mice per group. *p
Figure Legend Snippet: Lack of LDH-A results in higher levels of M1 Mø markers A–C . Mø were isolated from Cre tm -LDH-A flfl (ko) and LDH-A flfl (wt) mice injected with tamoxifen (10 mg/ml in corn oil; every day for 4 days; i.p.). Differentiated Mø were polarized with LPS (100 ng/ml) and IFNγ (20 ng/ml) (M1) or IL-4 (20 ng/ml) (M2) for 3–5 days, Western blotting with antibody against LDH-A is shown in A . The number of CD197 (CCR7)-PE + /CD86-APC + (M1) or MMR-PE + (M2) cells were measured by flow cytometry ( B C ). Data are representative for 3 independent experiments in triplicates. D–E . Flow cytometry with the antibodies against M1/M2-like skewing in Mø polarized in the presence of DMSO (vehicle) or LDH-A inhibitor (10 µM) for 3 days. n=3. F–G . Representative immunofluorescence staining of K-Ras lungs with antibody against iNOS and F4.80 are shown in F. Quantification is shown in G. Lungs were harvested after 10 days after induction of Cre with tamoxifen in K-Ras mice treated with doxycycline diet for 13 weeks to induce lung carcinogenesis prior tamoxifen. n=3–4 mice per group. *p

Techniques Used: Isolation, Mouse Assay, Injection, Western Blot, Flow Cytometry, Cytometry, Immunofluorescence, Staining

53) Product Images from "ISG15 governs mitochondrial function in macrophages following vaccinia virus infection"

Article Title: ISG15 governs mitochondrial function in macrophages following vaccinia virus infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006651

IFN and VACV infection increases proinflammatory cytokine levels in ISG15 -/- BMDM and increases arginase-1 activity. (A) ISG15 +/+ or ISG15 -/- BMDM were infected with VACV (1 PFU/cell). Cellular lysates collected at 2 and 6 hpi, or from mock-infected cultures, were analyzed by 12% SDS-PAGE, transferred to nitrocellulose membranes, and the expression of iNOS, Arg-1, ISG15 or β-actin (protein loading control) was examined by western blotting using specific antibodies. Uninfected M1 or M2 polarized ISG15 -/- BMDM were used as iNOS or Arg-1 controls. (B) Under the same conditions as above, the production of urea was measured as a marker of Arg-1 activity. The reaction was performed following the indications of the manufacturer. Results represent the mean ± the standard deviation of five biological replicates. (C) The expression level of TNF-α, IFN-β, IL-6, IL-1β and IL-12 genes was measured by quantitative RT-PCR. Triplicate samples were measured in three independent experiments; data shown is representative of one experiment. (D) IL-6 levels in the medium of ISG15 +/+ and ISG15 -/- BMDM were quantified by ELISA. Aliquots (100 μl) of supernatant from ISG15 +/+ or ISG15 -/- BMDM uninfected or at 2, 6, hpi were used for ELISA according to the manufacturer's instructions. Triplicate samples were measured in two independent experiments.
Figure Legend Snippet: IFN and VACV infection increases proinflammatory cytokine levels in ISG15 -/- BMDM and increases arginase-1 activity. (A) ISG15 +/+ or ISG15 -/- BMDM were infected with VACV (1 PFU/cell). Cellular lysates collected at 2 and 6 hpi, or from mock-infected cultures, were analyzed by 12% SDS-PAGE, transferred to nitrocellulose membranes, and the expression of iNOS, Arg-1, ISG15 or β-actin (protein loading control) was examined by western blotting using specific antibodies. Uninfected M1 or M2 polarized ISG15 -/- BMDM were used as iNOS or Arg-1 controls. (B) Under the same conditions as above, the production of urea was measured as a marker of Arg-1 activity. The reaction was performed following the indications of the manufacturer. Results represent the mean ± the standard deviation of five biological replicates. (C) The expression level of TNF-α, IFN-β, IL-6, IL-1β and IL-12 genes was measured by quantitative RT-PCR. Triplicate samples were measured in three independent experiments; data shown is representative of one experiment. (D) IL-6 levels in the medium of ISG15 +/+ and ISG15 -/- BMDM were quantified by ELISA. Aliquots (100 μl) of supernatant from ISG15 +/+ or ISG15 -/- BMDM uninfected or at 2, 6, hpi were used for ELISA according to the manufacturer's instructions. Triplicate samples were measured in two independent experiments.

Techniques Used: Infection, Activity Assay, SDS Page, Expressing, Western Blot, Marker, Standard Deviation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

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Centrifugation:

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MTT Assay:

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Article Snippet: Materials and Reagents GC (PubChem CID: 161120), Aspirin (PubChem CID: 2244), 2, 3, 5-Triphenyltetrazolium chloride (TTC), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and chloral hydrate were bought from Sigma (St. Louis, MO, United States). .. Anti-ICAM-1, anti-VCAM-1, anti-iNOS, anti-CD40, anti-NF-κB p65, anti-p-IκB-α, anti-IKK-β, anti-β-actin, anti-Histone, goat anti-rabbit and anti-mouse IgG antibodies were purchased from Santa Cruz (Santa Cruz, CA, United States).

Blocking Assay:

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Article Snippet: After transfer, the membranes were blocked with LI-COR blocking buffer for 45 min and incubated in primary antibodies following manufacturer’s protocol. .. The primary antibodies used are as follows: anti-caspase-1 (Adipogen, 1:1000) (AB_2490248), anti-NLRP3 (Adipogen, 1:1000), anti-iNOS (Santa Cruz, 1:1000), anti-IL-1β (R & D systems, 1:500), anti-Mfn2 (Cell Signaling, 1:1000), anti-VPS35 (Santa Cruz, 1:500), and anti-ASC (Adipogen, 1:1000) (AB_2490440).

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Article Snippet: .. After blocking, the membranes were incubated overnight at 4 °C with the primary antibodies including anti-occludin (1:1000; Invitrogen, Camarillo, CA), anti-COX-2 (1:1000; CST, Beverly, MA), anti-IL-1β, anti-iNOS (1:200; Santa Cruz Biotech, CA), anti-β-actin (1:500; ZSGB-Bio, Beijing, China) or anti-HMGB1 (1:1000; Proteintech, UK), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotech). .. Bands were detected by ECL advance Western blotting detection reagents (Millipore, Billercia, MA).

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Article Snippet: .. After blocking with Tris-buffered saline with Tween-20 [TBST; 20 mM Tris (pH 7.5), 150 mM NaCl and 0.01% Tween-20] containing 5% non-fat dry milk for 1 h at room temperature, the membranes were then incubated with polyclonal anti-DDAH-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal anti-eNOS (Sigma) and anti-iNOS (Santa Cruz Biotechnology) antibodies overnight at room temperature with constant agitation. .. The filters were then washed and probed with secondary HRP-linked goat anti-mouse or anti-rabbit antibodies (1:500; Santa Cruz Biotechnology) at room temperature for 1 h. The proteins were detected using an enhanced chemiluminescence (ECL) detection system.

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Article Snippet: .. Then, the membranes were incubated with anti-Bcl2 (Santa Cruz Biotechnology), anti-iNOS (Santa Cruz Biotechnology), and anti-NFκB-p65 antibodies (1:250) diluted in tris-buffered saline-tween containing 1% bovine serum albumin, and β actin (Santa Cruz Biotechnology) as internal control diluted 1:1000 in blocking buffer. .. Finally, images of indicated protein bands were recorded on the BioMax film (Kodak), and densitometrcally quantification was conducted by using Image J software (Bio-Rad, California, USA).

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Adsorption:

Article Title: Neuroprotective Effect of Artesunate in Experimental Model of Traumatic Brain Injury
Article Snippet: The sections were permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin, respectively. .. Afterwards, the sections were incubated overnight with one of the following primary antibodies diluted in PBS: polyclonal anti-glial cell line-derived neurotrophic factor (GDNF) (1:500, Santa Cruz Biotechnology), polyclonal anti-brain-derived neurotrophic factor (BDNF) (1:500, Santa Cruz Biotechnology), anti- IL-1β (1:500, Santa Cruz Biotechnology), anti-TNF-α (1:500, Santa Cruz Biotechnology), anti-iNOS. (1:500, Santa Cruz Biotechnology), anti-VEGF (1:500, Santa Cruz Biotechnology).

Electrophoresis:

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Article Snippet: Protein samples (30–80 μ g) were loaded on a 12% SDS-polyacrylamide gel and separated by electrophoresis prior to transfer to polyvinylidene difluoride membranes. .. After blocking with Tris-buffered saline with Tween-20 [TBST; 20 mM Tris (pH 7.5), 150 mM NaCl and 0.01% Tween-20] containing 5% non-fat dry milk for 1 h at room temperature, the membranes were then incubated with polyclonal anti-DDAH-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal anti-eNOS (Sigma) and anti-iNOS (Santa Cruz Biotechnology) antibodies overnight at room temperature with constant agitation.

Incubation:

Article Title: Neuroprotective Effect of Artesunate in Experimental Model of Traumatic Brain Injury
Article Snippet: .. Afterwards, the sections were incubated overnight with one of the following primary antibodies diluted in PBS: polyclonal anti-glial cell line-derived neurotrophic factor (GDNF) (1:500, Santa Cruz Biotechnology), polyclonal anti-brain-derived neurotrophic factor (BDNF) (1:500, Santa Cruz Biotechnology), anti- IL-1β (1:500, Santa Cruz Biotechnology), anti-TNF-α (1:500, Santa Cruz Biotechnology), anti-iNOS. (1:500, Santa Cruz Biotechnology), anti-VEGF (1:500, Santa Cruz Biotechnology). .. The immunohistochemical images were collected by Zeiss microscope using Axio Vision software.

Article Title: Manganese Activates NLRP3 Inflammasome Signaling and Propagates Exosomal Release of ASC in Microglial Cells
Article Snippet: Following primary antibody incubation, membranes were washed with PBS-TWEEN 20 (0.05%) for 1 h and incubated in LI-COR IR secondary antibodies for 1 h at room temperature, washed with PBS-TWEEN for 1 h, and imaged using an Odyssey scanner. .. The primary antibodies used are as follows: anti-caspase-1 (Adipogen, 1:1000) (AB_2490248), anti-NLRP3 (Adipogen, 1:1000), anti-iNOS (Santa Cruz, 1:1000), anti-IL-1β (R & D systems, 1:500), anti-Mfn2 (Cell Signaling, 1:1000), anti-VPS35 (Santa Cruz, 1:500), and anti-ASC (Adipogen, 1:1000) (AB_2490440).

Article Title: Cell permeable HMGB1-binding heptamer peptide ameliorates neurovascular complications associated with thrombolytic therapy in rats with transient ischemic stroke
Article Snippet: .. After blocking, the membranes were incubated overnight at 4 °C with the primary antibodies including anti-occludin (1:1000; Invitrogen, Camarillo, CA), anti-COX-2 (1:1000; CST, Beverly, MA), anti-IL-1β, anti-iNOS (1:200; Santa Cruz Biotech, CA), anti-β-actin (1:500; ZSGB-Bio, Beijing, China) or anti-HMGB1 (1:1000; Proteintech, UK), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotech). .. Bands were detected by ECL advance Western blotting detection reagents (Millipore, Billercia, MA).

Article Title: Ghrelin protects against contact dermatitis and psoriasiform skin inflammation by antagonizing TNF-α/NF-κB signaling pathways
Article Snippet: .. Immunohistochemistry Tissues from each experimental group of the 2 models were collected, fixed for 48 hours in 10% formalin and then prepared and incubated with polyclonal anti-CD4, anti-CD68, anti-iNOS, anti-p-IκBα and anti-ghrelin serum (anti-CD4 antibody: 1:100 dilution, sc-70670, Santa Cruz Biotechnology, U.S.A.; anti-CD68 antibody: 1:100 dilution, sc-7084, Santa Cruz Biotechnology, U.S.A.; anti-iNOS antibody: 1:150 dilution, 18985-1-AP, Proteintech Biotechnology, U.S.A.; p-IκBα: 1:100 dilution, sc-101713, Santa Cruz, U.S.A.; anti-ghrelin antibody: 1:100 dilution, sc50297, Santa Cruz Biotechnology, U.S.A. ) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Zhongshanjinqiao Biotechnology, P.R., China) for 60 minutes at RT. .. Signal was detected by using the Vector Elite ABC Kit (Vectastain; Vector).

Article Title: High glucose enhances LPS-stimulated human PMVEC hyperpermeability via the NO pathway
Article Snippet: .. After blocking with Tris-buffered saline with Tween-20 [TBST; 20 mM Tris (pH 7.5), 150 mM NaCl and 0.01% Tween-20] containing 5% non-fat dry milk for 1 h at room temperature, the membranes were then incubated with polyclonal anti-DDAH-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal anti-eNOS (Sigma) and anti-iNOS (Santa Cruz Biotechnology) antibodies overnight at room temperature with constant agitation. .. The filters were then washed and probed with secondary HRP-linked goat anti-mouse or anti-rabbit antibodies (1:500; Santa Cruz Biotechnology) at room temperature for 1 h. The proteins were detected using an enhanced chemiluminescence (ECL) detection system.

Article Title: Inhibition of TNF in the Brain Reverses Alterations in RAS Components and Attenuates Angiotensin II-Induced Hypertension
Article Snippet: The membranes were subjected to immunoblot analyses with anti-ACE (Santa Cruz, 1∶500), anti-AT1 R (Santa Cruz, 1∶500), anti-ACE2 (Santa Cruz, 1∶500), anti-AT2 R (Santa Cruz, 1∶200), anti-Mas (Alomone Labs, 1∶500), anti-NOX-2 (BD Biosciences, 1∶500), anti-NOX-4 (Santa Cruz, 1∶1000), anti-iNOS (Santa Cruz, 1∶500), anti-nNOS (Santa Cruz, 1∶500), and anti-GAPDH (Santa Cruz, 1∶1000) antibodies. .. The membranes were washed and incubated with anti-rabbit or anti-goat secondary antibodies (Santa Cruz, 1∶5000) for 1 hour at room temperature.

Article Title: Ameliorative effect of taurine-chloramine in azathioprine-induced testicular damage; a deeper insight into the mechanism of protection
Article Snippet: .. Then, the membranes were incubated with anti-Bcl2 (Santa Cruz Biotechnology), anti-iNOS (Santa Cruz Biotechnology), and anti-NFκB-p65 antibodies (1:250) diluted in tris-buffered saline-tween containing 1% bovine serum albumin, and β actin (Santa Cruz Biotechnology) as internal control diluted 1:1000 in blocking buffer. .. Finally, images of indicated protein bands were recorded on the BioMax film (Kodak), and densitometrcally quantification was conducted by using Image J software (Bio-Rad, California, USA).

Article Title: 12/15-Lipoxygenase metabolites of arachidonic acid activate PPARγ: a possible neuroprotective effect in ischemic brain
Article Snippet: After blocking for 1 h in 0.1% Tween 20/PBS (PBS-T) containing 5% fat-free milk, the blot was incubated with the primary antibody at 4°C overnight. .. The antibodies were anti-PPARγ (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-12/15-LOX (1:1,000 dilution; Cayman Chemical), anti-COX-2 (1:1,000 dilution; Santa Cruz Biotechnology), or anti-iNOS (1:200 dilution; Santa Cruz Biotechnology).

Expressing:

Article Title: Inhibition of TNF in the Brain Reverses Alterations in RAS Components and Attenuates Angiotensin II-Induced Hypertension
Article Snippet: The membranes were subjected to immunoblot analyses with anti-ACE (Santa Cruz, 1∶500), anti-AT1 R (Santa Cruz, 1∶500), anti-ACE2 (Santa Cruz, 1∶500), anti-AT2 R (Santa Cruz, 1∶200), anti-Mas (Alomone Labs, 1∶500), anti-NOX-2 (BD Biosciences, 1∶500), anti-NOX-4 (Santa Cruz, 1∶1000), anti-iNOS (Santa Cruz, 1∶500), anti-nNOS (Santa Cruz, 1∶500), and anti-GAPDH (Santa Cruz, 1∶1000) antibodies. .. The bands were quantified by densitometry using Chemidoc XRS system and Quantity-One software (Bio-Rad) and were normalized to GAPDH expression.

Modification:

Article Title: Isolation of Novel Sesquiterpeniods and Anti-neuroinflammatory Metabolites from Nardostachys jatamansi
Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco BRL Co. (Grand Island, NY, USA). .. 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 (Dallas, TX, USA) [ ].

Western Blot:

Article Title: Manganese Activates NLRP3 Inflammasome Signaling and Propagates Exosomal Release of ASC in Microglial Cells
Article Snippet: Paragraph title: Western blot ... The primary antibodies used are as follows: anti-caspase-1 (Adipogen, 1:1000) (AB_2490248), anti-NLRP3 (Adipogen, 1:1000), anti-iNOS (Santa Cruz, 1:1000), anti-IL-1β (R & D systems, 1:500), anti-Mfn2 (Cell Signaling, 1:1000), anti-VPS35 (Santa Cruz, 1:500), and anti-ASC (Adipogen, 1:1000) (AB_2490440).

Article Title: Cell permeable HMGB1-binding heptamer peptide ameliorates neurovascular complications associated with thrombolytic therapy in rats with transient ischemic stroke
Article Snippet: Paragraph title: Western blot analysis ... After blocking, the membranes were incubated overnight at 4 °C with the primary antibodies including anti-occludin (1:1000; Invitrogen, Camarillo, CA), anti-COX-2 (1:1000; CST, Beverly, MA), anti-IL-1β, anti-iNOS (1:200; Santa Cruz Biotech, CA), anti-β-actin (1:500; ZSGB-Bio, Beijing, China) or anti-HMGB1 (1:1000; Proteintech, UK), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotech).

Article Title: Celastrol Prevents Atherosclerosis via Inhibiting LOX-1 and Oxidative Stress
Article Snippet: .. Western blot analysis Cytoplasmic and nuclear protein samples were separated on 10% v/v sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, USA) and then immunoblotted with primary anti-NF-κB p65, anti-LOX-1 (1∶1000, Abcam), anti-IκBα, anti-p-IκBα, anti-iNOS, anti-p47-phox(1∶1000, Santa Cruz Biotechnology), anti-α-tublin (1∶1000) or anti-β-actin(1∶2000, Cell Signaling Technology). .. Protein were visualized by enhanced chemiluminescence substrate (Thermo).

Article Title: High glucose enhances LPS-stimulated human PMVEC hyperpermeability via the NO pathway
Article Snippet: Paragraph title: Western blotting ... After blocking with Tris-buffered saline with Tween-20 [TBST; 20 mM Tris (pH 7.5), 150 mM NaCl and 0.01% Tween-20] containing 5% non-fat dry milk for 1 h at room temperature, the membranes were then incubated with polyclonal anti-DDAH-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal anti-eNOS (Sigma) and anti-iNOS (Santa Cruz Biotechnology) antibodies overnight at room temperature with constant agitation.

Article Title: Inhibition of TNF in the Brain Reverses Alterations in RAS Components and Attenuates Angiotensin II-Induced Hypertension
Article Snippet: Paragraph title: Western Blot Analysis ... The membranes were subjected to immunoblot analyses with anti-ACE (Santa Cruz, 1∶500), anti-AT1 R (Santa Cruz, 1∶500), anti-ACE2 (Santa Cruz, 1∶500), anti-AT2 R (Santa Cruz, 1∶200), anti-Mas (Alomone Labs, 1∶500), anti-NOX-2 (BD Biosciences, 1∶500), anti-NOX-4 (Santa Cruz, 1∶1000), anti-iNOS (Santa Cruz, 1∶500), anti-nNOS (Santa Cruz, 1∶500), and anti-GAPDH (Santa Cruz, 1∶1000) antibodies.

Article Title: Ameliorative effect of taurine-chloramine in azathioprine-induced testicular damage; a deeper insight into the mechanism of protection
Article Snippet: Paragraph title: Western blotting assays ... Then, the membranes were incubated with anti-Bcl2 (Santa Cruz Biotechnology), anti-iNOS (Santa Cruz Biotechnology), and anti-NFκB-p65 antibodies (1:250) diluted in tris-buffered saline-tween containing 1% bovine serum albumin, and β actin (Santa Cruz Biotechnology) as internal control diluted 1:1000 in blocking buffer.

Article Title: 12/15-Lipoxygenase metabolites of arachidonic acid activate PPARγ: a possible neuroprotective effect in ischemic brain
Article Snippet: Paragraph title: Western blot analysis ... The antibodies were anti-PPARγ (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-12/15-LOX (1:1,000 dilution; Cayman Chemical), anti-COX-2 (1:1,000 dilution; Santa Cruz Biotechnology), or anti-iNOS (1:200 dilution; Santa Cruz Biotechnology).

Flow Cytometry:

Article Title: Isolation of Novel Sesquiterpeniods and Anti-neuroinflammatory Metabolites from Nardostachys jatamansi
Article Snippet: HPLC separations were performed on a prep-C18 column (21.2 × 150 mm; 5 μm particle size) with a flow rate of 5 mL/min, and a semiprep-C18 column (10 × 250 mm; 5 μm particle size) with a flow rate of 3 mL/min. .. 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 (Dallas, TX, USA) [ ].

High Performance Liquid Chromatography:

Article Title: Isolation of Novel Sesquiterpeniods and Anti-neuroinflammatory Metabolites from Nardostachys jatamansi
Article Snippet: HPLC separations were performed on a prep-C18 column (21.2 × 150 mm; 5 μm particle size) with a flow rate of 5 mL/min, and a semiprep-C18 column (10 × 250 mm; 5 μm particle size) with a flow rate of 3 mL/min. .. 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 (Dallas, TX, USA) [ ].

Immunohistochemistry:

Article Title: Neuroprotective Effect of Artesunate in Experimental Model of Traumatic Brain Injury
Article Snippet: Paragraph title: Immunohistochemistry ... Afterwards, the sections were incubated overnight with one of the following primary antibodies diluted in PBS: polyclonal anti-glial cell line-derived neurotrophic factor (GDNF) (1:500, Santa Cruz Biotechnology), polyclonal anti-brain-derived neurotrophic factor (BDNF) (1:500, Santa Cruz Biotechnology), anti- IL-1β (1:500, Santa Cruz Biotechnology), anti-TNF-α (1:500, Santa Cruz Biotechnology), anti-iNOS. (1:500, Santa Cruz Biotechnology), anti-VEGF (1:500, Santa Cruz Biotechnology).

Article Title: Ghrelin protects against contact dermatitis and psoriasiform skin inflammation by antagonizing TNF-α/NF-κB signaling pathways
Article Snippet: .. Immunohistochemistry Tissues from each experimental group of the 2 models were collected, fixed for 48 hours in 10% formalin and then prepared and incubated with polyclonal anti-CD4, anti-CD68, anti-iNOS, anti-p-IκBα and anti-ghrelin serum (anti-CD4 antibody: 1:100 dilution, sc-70670, Santa Cruz Biotechnology, U.S.A.; anti-CD68 antibody: 1:100 dilution, sc-7084, Santa Cruz Biotechnology, U.S.A.; anti-iNOS antibody: 1:150 dilution, 18985-1-AP, Proteintech Biotechnology, U.S.A.; p-IκBα: 1:100 dilution, sc-101713, Santa Cruz, U.S.A.; anti-ghrelin antibody: 1:100 dilution, sc50297, Santa Cruz Biotechnology, U.S.A. ) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Zhongshanjinqiao Biotechnology, P.R., China) for 60 minutes at RT. .. Signal was detected by using the Vector Elite ABC Kit (Vectastain; Vector).

Protease Inhibitor:

Article Title: HemoHIM, a herbal preparation, alleviates airway inflammation caused by cigarette smoke and lipopolysaccharide
Article Snippet: Immunoblotting The lung tissue was homogenized (1/10 w/v) using a homogenizer in a Tissue Lysis/Extraction reagent (Sigma-Aldrich, St, Louis, MO, USA) that contained a protease inhibitor cocktail (Sigma-Aldrich). .. The following primary antibodies and dilutions were used: anti-β-actin (1:2000 dilution; Cell Signaling, Danvers, MA, USA), anti-pERK (1:1000 dilution; Cell Signaling), anti-ERK (1:1000 dilution; Cell Signaling) and anti-iNOS (1:1000 dilution; Santa Cruz Biotechnology, MA, USA).

Protein Concentration:

Article Title: High glucose enhances LPS-stimulated human PMVEC hyperpermeability via the NO pathway
Article Snippet: The total protein concentration was determined using a Protein Assay kit (Bio-Rad, Hercules, CA, USA). .. After blocking with Tris-buffered saline with Tween-20 [TBST; 20 mM Tris (pH 7.5), 150 mM NaCl and 0.01% Tween-20] containing 5% non-fat dry milk for 1 h at room temperature, the membranes were then incubated with polyclonal anti-DDAH-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal anti-eNOS (Sigma) and anti-iNOS (Santa Cruz Biotechnology) antibodies overnight at room temperature with constant agitation.

Article Title: Inhibition of TNF in the Brain Reverses Alterations in RAS Components and Attenuates Angiotensin II-Induced Hypertension
Article Snippet: The protein concentration was measured using a bicinchioninic acid protein assay kit (Pierce). .. The membranes were subjected to immunoblot analyses with anti-ACE (Santa Cruz, 1∶500), anti-AT1 R (Santa Cruz, 1∶500), anti-ACE2 (Santa Cruz, 1∶500), anti-AT2 R (Santa Cruz, 1∶200), anti-Mas (Alomone Labs, 1∶500), anti-NOX-2 (BD Biosciences, 1∶500), anti-NOX-4 (Santa Cruz, 1∶1000), anti-iNOS (Santa Cruz, 1∶500), anti-nNOS (Santa Cruz, 1∶500), and anti-GAPDH (Santa Cruz, 1∶1000) antibodies.

Binding Assay:

Article Title: Neuroprotective Effect of Artesunate in Experimental Model of Traumatic Brain Injury
Article Snippet: The sections were permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin, respectively. .. Afterwards, the sections were incubated overnight with one of the following primary antibodies diluted in PBS: polyclonal anti-glial cell line-derived neurotrophic factor (GDNF) (1:500, Santa Cruz Biotechnology), polyclonal anti-brain-derived neurotrophic factor (BDNF) (1:500, Santa Cruz Biotechnology), anti- IL-1β (1:500, Santa Cruz Biotechnology), anti-TNF-α (1:500, Santa Cruz Biotechnology), anti-iNOS. (1:500, Santa Cruz Biotechnology), anti-VEGF (1:500, Santa Cruz Biotechnology).

Article Title: Microglia and motor neurons during disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis: changes in arginase1 and inducible nitric oxide synthase
Article Snippet: .. The following primary antibodies were used: anti-ionized calcium binding adaptor molecule 1 (Iba1, Wako, Osaka, Japan); anti-iNOS (Santa Cruz Biotechnology, Dallas, TX, USA); anti-Arg1 (Santa Cruz Biotechnology); anti-ubiquitin (Dako, Carpinteria, CA, USA); and anti-dephosphorylated neurofilament heavy and medium chain (SMI32, Covance, Princeton, NJ, USA) (Table ). .. AlexaFluor-conjugated and biotin-conjugated secondary antibodies, AlexaFluor-conjugated streptavidin, and Nuclear Yellow dye were obtained from Molecular Probes (Life Technologies, Carlsbad, CA, USA) (Table ).

Nucleic Acid Electrophoresis:

Article Title: HemoHIM, a herbal preparation, alleviates airway inflammation caused by cigarette smoke and lipopolysaccharide
Article Snippet: Equal amounts of the total protein (30 µg) were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. .. The following primary antibodies and dilutions were used: anti-β-actin (1:2000 dilution; Cell Signaling, Danvers, MA, USA), anti-pERK (1:1000 dilution; Cell Signaling), anti-ERK (1:1000 dilution; Cell Signaling) and anti-iNOS (1:1000 dilution; Santa Cruz Biotechnology, MA, USA).

Avidin-Biotin Assay:

Article Title: Neuroprotective Effect of Artesunate in Experimental Model of Traumatic Brain Injury
Article Snippet: The sections were permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin, respectively. .. Afterwards, the sections were incubated overnight with one of the following primary antibodies diluted in PBS: polyclonal anti-glial cell line-derived neurotrophic factor (GDNF) (1:500, Santa Cruz Biotechnology), polyclonal anti-brain-derived neurotrophic factor (BDNF) (1:500, Santa Cruz Biotechnology), anti- IL-1β (1:500, Santa Cruz Biotechnology), anti-TNF-α (1:500, Santa Cruz Biotechnology), anti-iNOS. (1:500, Santa Cruz Biotechnology), anti-VEGF (1:500, Santa Cruz Biotechnology).

Microscopy:

Article Title: Neuroprotective Effect of Artesunate in Experimental Model of Traumatic Brain Injury
Article Snippet: Afterwards, the sections were incubated overnight with one of the following primary antibodies diluted in PBS: polyclonal anti-glial cell line-derived neurotrophic factor (GDNF) (1:500, Santa Cruz Biotechnology), polyclonal anti-brain-derived neurotrophic factor (BDNF) (1:500, Santa Cruz Biotechnology), anti- IL-1β (1:500, Santa Cruz Biotechnology), anti-TNF-α (1:500, Santa Cruz Biotechnology), anti-iNOS. (1:500, Santa Cruz Biotechnology), anti-VEGF (1:500, Santa Cruz Biotechnology). .. The immunohistochemical images were collected by Zeiss microscope using Axio Vision software.

Polyacrylamide Gel Electrophoresis:

Article Title: Celastrol Prevents Atherosclerosis via Inhibiting LOX-1 and Oxidative Stress
Article Snippet: .. Western blot analysis Cytoplasmic and nuclear protein samples were separated on 10% v/v sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, USA) and then immunoblotted with primary anti-NF-κB p65, anti-LOX-1 (1∶1000, Abcam), anti-IκBα, anti-p-IκBα, anti-iNOS, anti-p47-phox(1∶1000, Santa Cruz Biotechnology), anti-α-tublin (1∶1000) or anti-β-actin(1∶2000, Cell Signaling Technology). .. Protein were visualized by enhanced chemiluminescence substrate (Thermo).

Staining:

Article Title: Neuroprotective Effect of Artesunate in Experimental Model of Traumatic Brain Injury
Article Snippet: Afterwards, the sections were incubated overnight with one of the following primary antibodies diluted in PBS: polyclonal anti-glial cell line-derived neurotrophic factor (GDNF) (1:500, Santa Cruz Biotechnology), polyclonal anti-brain-derived neurotrophic factor (BDNF) (1:500, Santa Cruz Biotechnology), anti- IL-1β (1:500, Santa Cruz Biotechnology), anti-TNF-α (1:500, Santa Cruz Biotechnology), anti-iNOS. (1:500, Santa Cruz Biotechnology), anti-VEGF (1:500, Santa Cruz Biotechnology). .. For graphic representation of densitometric analyses, we measured the intensity of positive staining (brown staining) by computer-assisted color image analysis (Leica QWin V3, UK).

SDS Page:

Article Title: Celastrol Prevents Atherosclerosis via Inhibiting LOX-1 and Oxidative Stress
Article Snippet: .. Western blot analysis Cytoplasmic and nuclear protein samples were separated on 10% v/v sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, USA) and then immunoblotted with primary anti-NF-κB p65, anti-LOX-1 (1∶1000, Abcam), anti-IκBα, anti-p-IκBα, anti-iNOS, anti-p47-phox(1∶1000, Santa Cruz Biotechnology), anti-α-tublin (1∶1000) or anti-β-actin(1∶2000, Cell Signaling Technology). .. Protein were visualized by enhanced chemiluminescence substrate (Thermo).

Article Title: Inhibition of TNF in the Brain Reverses Alterations in RAS Components and Attenuates Angiotensin II-Induced Hypertension
Article Snippet: Equal amounts of protein (5 µg) were separated by SDS-PAGE on 10% or 12% gels, transferred on to PVDF membrane (Immobilon-P, Millipore), and blocked with 1% BSA in TBS-T at room temperature for 60 min. .. The membranes were subjected to immunoblot analyses with anti-ACE (Santa Cruz, 1∶500), anti-AT1 R (Santa Cruz, 1∶500), anti-ACE2 (Santa Cruz, 1∶500), anti-AT2 R (Santa Cruz, 1∶200), anti-Mas (Alomone Labs, 1∶500), anti-NOX-2 (BD Biosciences, 1∶500), anti-NOX-4 (Santa Cruz, 1∶1000), anti-iNOS (Santa Cruz, 1∶500), anti-nNOS (Santa Cruz, 1∶500), and anti-GAPDH (Santa Cruz, 1∶1000) antibodies.

Article Title: Myeloid cell-derived inducible nitric oxide synthase suppresses M1 macrophage polarization
Article Snippet: Cell lysates were clarified by centrifugation (4 °C, 15 min, 14,000 r.p.m.) and protein was subjected to 10% sodium dodecyl sulfate–PAGE (SDS–PAGE) and immunoblotting was performed. .. Anti-iNOS (Santa Cruz), anti-IRF5 (MBL), anti-STAT1 and anti-β-actin (Sigma) antibodies were used according to the manufactures’ instructions.

Article Title: Ameliorative effect of taurine-chloramine in azathioprine-induced testicular damage; a deeper insight into the mechanism of protection
Article Snippet: Samples of equal protein concentrations were electrophoresed using 10% SDS/PAGE and electro-transferred to polyvinylidene difluoride membranes. .. Then, the membranes were incubated with anti-Bcl2 (Santa Cruz Biotechnology), anti-iNOS (Santa Cruz Biotechnology), and anti-NFκB-p65 antibodies (1:250) diluted in tris-buffered saline-tween containing 1% bovine serum albumin, and β actin (Santa Cruz Biotechnology) as internal control diluted 1:1000 in blocking buffer.

Article Title: 12/15-Lipoxygenase metabolites of arachidonic acid activate PPARγ: a possible neuroprotective effect in ischemic brain
Article Snippet: Proteins were separated on 8% SDS-PAGE, and then transferred onto nitrocellulose membrane. .. The antibodies were anti-PPARγ (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-12/15-LOX (1:1,000 dilution; Cayman Chemical), anti-COX-2 (1:1,000 dilution; Santa Cruz Biotechnology), or anti-iNOS (1:200 dilution; Santa Cruz Biotechnology).

Plasmid Preparation:

Article Title: Ghrelin protects against contact dermatitis and psoriasiform skin inflammation by antagonizing TNF-α/NF-κB signaling pathways
Article Snippet: Immunohistochemistry Tissues from each experimental group of the 2 models were collected, fixed for 48 hours in 10% formalin and then prepared and incubated with polyclonal anti-CD4, anti-CD68, anti-iNOS, anti-p-IκBα and anti-ghrelin serum (anti-CD4 antibody: 1:100 dilution, sc-70670, Santa Cruz Biotechnology, U.S.A.; anti-CD68 antibody: 1:100 dilution, sc-7084, Santa Cruz Biotechnology, U.S.A.; anti-iNOS antibody: 1:150 dilution, 18985-1-AP, Proteintech Biotechnology, U.S.A.; p-IκBα: 1:100 dilution, sc-101713, Santa Cruz, U.S.A.; anti-ghrelin antibody: 1:100 dilution, sc50297, Santa Cruz Biotechnology, U.S.A. ) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Zhongshanjinqiao Biotechnology, P.R., China) for 60 minutes at RT. .. Signal was detected by using the Vector Elite ABC Kit (Vectastain; Vector).

Software:

Article Title: Neuroprotective Effect of Artesunate in Experimental Model of Traumatic Brain Injury
Article Snippet: Afterwards, the sections were incubated overnight with one of the following primary antibodies diluted in PBS: polyclonal anti-glial cell line-derived neurotrophic factor (GDNF) (1:500, Santa Cruz Biotechnology), polyclonal anti-brain-derived neurotrophic factor (BDNF) (1:500, Santa Cruz Biotechnology), anti- IL-1β (1:500, Santa Cruz Biotechnology), anti-TNF-α (1:500, Santa Cruz Biotechnology), anti-iNOS. (1:500, Santa Cruz Biotechnology), anti-VEGF (1:500, Santa Cruz Biotechnology). .. The immunohistochemical images were collected by Zeiss microscope using Axio Vision software.

Article Title: Inhibition of TNF in the Brain Reverses Alterations in RAS Components and Attenuates Angiotensin II-Induced Hypertension
Article Snippet: The membranes were subjected to immunoblot analyses with anti-ACE (Santa Cruz, 1∶500), anti-AT1 R (Santa Cruz, 1∶500), anti-ACE2 (Santa Cruz, 1∶500), anti-AT2 R (Santa Cruz, 1∶200), anti-Mas (Alomone Labs, 1∶500), anti-NOX-2 (BD Biosciences, 1∶500), anti-NOX-4 (Santa Cruz, 1∶1000), anti-iNOS (Santa Cruz, 1∶500), anti-nNOS (Santa Cruz, 1∶500), and anti-GAPDH (Santa Cruz, 1∶1000) antibodies. .. The bands were quantified by densitometry using Chemidoc XRS system and Quantity-One software (Bio-Rad) and were normalized to GAPDH expression.

Article Title: Ameliorative effect of taurine-chloramine in azathioprine-induced testicular damage; a deeper insight into the mechanism of protection
Article Snippet: Then, the membranes were incubated with anti-Bcl2 (Santa Cruz Biotechnology), anti-iNOS (Santa Cruz Biotechnology), and anti-NFκB-p65 antibodies (1:250) diluted in tris-buffered saline-tween containing 1% bovine serum albumin, and β actin (Santa Cruz Biotechnology) as internal control diluted 1:1000 in blocking buffer. .. Finally, images of indicated protein bands were recorded on the BioMax film (Kodak), and densitometrcally quantification was conducted by using Image J software (Bio-Rad, California, USA).

Enzyme-linked Immunosorbent Assay:

Article Title: Ginkgolide C Alleviates Myocardial Ischemia/Reperfusion-Induced Inflammatory Injury via Inhibition of CD40-NF-κB Pathway
Article Snippet: TNF-α, IL-1β and IL-6 ELISA kits were bought from Sigma (St. Louis, MO, United States). .. Anti-ICAM-1, anti-VCAM-1, anti-iNOS, anti-CD40, anti-NF-κB p65, anti-p-IκB-α, anti-IKK-β, anti-β-actin, anti-Histone, goat anti-rabbit and anti-mouse IgG antibodies were purchased from Santa Cruz (Santa Cruz, CA, United States).

Column Chromatography:

Article Title: Isolation of Novel Sesquiterpeniods and Anti-neuroinflammatory Metabolites from Nardostachys jatamansi
Article Snippet: Flash column chromatography was carried out using octadecyl-functionalized C18 silica gel (YMC, Kyoto, Japan) and silica gel (Merck, Kenilworth, NJ, USA). .. 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 (Dallas, TX, USA) [ ].

Lysis:

Article Title: HemoHIM, a herbal preparation, alleviates airway inflammation caused by cigarette smoke and lipopolysaccharide
Article Snippet: Immunoblotting The lung tissue was homogenized (1/10 w/v) using a homogenizer in a Tissue Lysis/Extraction reagent (Sigma-Aldrich, St, Louis, MO, USA) that contained a protease inhibitor cocktail (Sigma-Aldrich). .. The following primary antibodies and dilutions were used: anti-β-actin (1:2000 dilution; Cell Signaling, Danvers, MA, USA), anti-pERK (1:1000 dilution; Cell Signaling), anti-ERK (1:1000 dilution; Cell Signaling) and anti-iNOS (1:1000 dilution; Santa Cruz Biotechnology, MA, USA).

Article Title: Inhibition of TNF in the Brain Reverses Alterations in RAS Components and Attenuates Angiotensin II-Induced Hypertension
Article Snippet: The PVN tissue was homogenized with RIPA lysis buffer. .. The membranes were subjected to immunoblot analyses with anti-ACE (Santa Cruz, 1∶500), anti-AT1 R (Santa Cruz, 1∶500), anti-ACE2 (Santa Cruz, 1∶500), anti-AT2 R (Santa Cruz, 1∶200), anti-Mas (Alomone Labs, 1∶500), anti-NOX-2 (BD Biosciences, 1∶500), anti-NOX-4 (Santa Cruz, 1∶1000), anti-iNOS (Santa Cruz, 1∶500), anti-nNOS (Santa Cruz, 1∶500), and anti-GAPDH (Santa Cruz, 1∶1000) antibodies.

Article Title: Myeloid cell-derived inducible nitric oxide synthase suppresses M1 macrophage polarization
Article Snippet: Immunoblotting analysis Cells were washed with cold phosphate-buffered saline and lysed for 15 min on ice in 0.5 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 280 mM NaCl, 0.5% Nonidet P-40, 0.2 mM EDTA, 2 mM EGTA, 10% glycerol and 1 mM dithiothreitol) containing protease inhibitors. .. Anti-iNOS (Santa Cruz), anti-IRF5 (MBL), anti-STAT1 and anti-β-actin (Sigma) antibodies were used according to the manufactures’ instructions.

Article Title: Ameliorative effect of taurine-chloramine in azathioprine-induced testicular damage; a deeper insight into the mechanism of protection
Article Snippet: Testicles samples were homogenized in ice-cold lysis buffer, and the homogenates were centrifuged at 14,000×g for 20 min at 4 °C. .. Then, the membranes were incubated with anti-Bcl2 (Santa Cruz Biotechnology), anti-iNOS (Santa Cruz Biotechnology), and anti-NFκB-p65 antibodies (1:250) diluted in tris-buffered saline-tween containing 1% bovine serum albumin, and β actin (Santa Cruz Biotechnology) as internal control diluted 1:1000 in blocking buffer.

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  • 99
    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.
    Anti Inos, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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