PA1-036

antibody from Invitrogen Antibodies

Targeting: NOS2 HEP-NOS, iNOS, NOS, NOS2A

Provider product page for PA1-036

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [133]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [2]
Immunohistochemistry [2]
Other assay [39]

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Product number: PA1-036 - Provider product page
Provider: Invitrogen Antibodies
Product name: iNOS Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-036 detects inducible nitric oxide synthase (iNOS) from human, mouse and rat tissues and cells as well as recombinant human and mouse iNOS. This antibody does not detect other NOS isoforms.
Concentration: 4 mg/mL

NOS2 protein structure - PA1-036 shown in red.

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Experimental details: Western blot analysis of iNOS was performed by loading 20 µg of RAW264 whole cell lysate untreated (left lane) or stimulated with LPS at 1 µg/mL for 16 hours (right lane) and 10 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) onto a 4-20% Tris-Glycine polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 5% Milk in TBST for at least 1 hour. The membrane was probed with an iNOS Rabbit polyclonal antibody (Product # PA1-036) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBST, and probed with a Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036) at a dilution of 1:1000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34087).

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Experimental details: Western blot was performed using Anti-iNOS Polyclonal Antibody (Product # PA1-036) and a 130kDa band corresponding to Nitric oxide synthase, inducible, was observed to be upregulated in RAW 264.7 cells upon treatment with LPS. Whole cell extracts (30 µg lysate) of RAW 264.7 (Lane 1) and RAW 264.7 treated with LPS (Lane 2) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). An uncharacterized band (*) was also observed at ~60kDa.

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Experimental details: Immunohistochemistry was performed on normal deparaffinized human Heart tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a rabbit polyclonal antibody recognizing iNOS (Product # PA1-036) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Immunohistochemistry was performed on normal deparaffinized human Lung tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a rabbit polyclonal antibody recognizing iNOS (Product # PA1-036) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Figure 4 Cerium oxide (CeO 2 ) nanoparticle treatment attenuates diaphragmatic inflammation after septic insult. Notes: Cecal inoculum (CI) induced increases in Stat3 phosphorylation are diminished with nanoparticle treatment ( A , n=6 rats/each group). Real-time PCR showed that iNOS gene expression levels was increased with CI group and diminished with nanoparticle treatment at 18 hours ( B , n=6 rats/each group). Immunoblot for iNOS/alpha-tubulin is shown ( C ). *vs Control, **vs CI: P

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Experimental details: Figure 5 Absence of epithelial Stat3 activity increases iNOS expression during involution. (A) Expression of inducible nitric oxide synthetase (iNOS) at 96 h of involution in control and Stat3 KO mice, measured by qRT-PCR relative to expression of cyclophilin (a housekeeping gene); values are mean +- SD from at least three experimental repeats, with each bar representing an individual mouse; * p < 0.05, as determined by Student's t -test. (B) Immunofluorescence staining for iNOS (red), E-cadherin (green) and DNA (Hoechst; blue) at 96 h of involution on control and Stat3 KO tissue. Arrows indicate cytoplasmic iNOS expression within cells with morphology consistent with macrophages; scale bars = 100 um

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Experimental details: Fig 2 iNOS protein is recalcitrant to IL-1beta-induction in canine articular chondrocytes. (A) iNOS mRNA expression in chondrocytes (P1) stimulated with 20 ng/mL of rhIL-1beta for 0, 3, 6, 9 and 12 hr, (B) 20 mug protein from each treatment time was denatured and separated on a 12% SDS page and silver stained to visualize the profile of separated proteins, (C) iNOS protein levels were undetectable by Western blot, an indication of resistance to cytokine induction. L; 100bp DNA ladder, ES; iNOS Electrophoresis Standard ~130 kDa (loaded at 100 ng). M; protein standards marker, results shown are from one representative experiment of three (n = 3) independent experiments.

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Experimental details: Fig 3 iNOS protein is recalcitrant to single or multiple cytokine induction. iNOS protein levels were undetectable in chondrocytes stimulated with single rcIL-1beta, TNF-alpha or LPS. A combination of rcIL-1beta + TNF-alpha + LPS induced a strong iNOS protein expression while a combination of TNF-alpha + LPS induced a very weak iNOS protein band as indicated by black arrowheads. HIF-1alpha and HIF-2alpha proteins were undetectable. ES; iNOS Standard protein ~130 kDa (loaded at 500 ng), M; Protein Standards marker. F-Actin (Internal control): 42 kDa, iNOS: 130 kDa, HIF-1alpha: 120 kDa and HIF-2alpha: 115 kDa. Blots shown are from one representative experiment of three (n = 3) independent experiments.

Supportive validation

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Experimental details: Fig 4 PPS inhibits rcIL-1beta + TNF-alpha + LPS-induced iNOS protein expression. Single rcIL-1beta (10 ng/mL) induced a weak iNOS protein signal compared to a combination of rcIL-1beta (10 ng/mL) + TNF-alpha (10 ng/mL) + LPS (50 mug/mL) which significantly induced iNOS protein expression in canine chondrocytes. Pre-incubation of canine chondrocytes with 40 mug/mL of PPS significantly inhibited rcIL-1beta + TNF-alpha + LPS-induced iNOS protein expression. M; Protein Standards marker. F-Actin (Internal control): 42 kDa, iNOS: 130 kDa. Blots shown are from two representative experiments of three (3) experiments.

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Experimental details: 3 FIGURE Representative photomicrographs illustrating the morphology exhibited by microglia in the medial preoptic nucleus of developing and adult Japanese quail. Ameboid microglia (black arrowheads) and transitioning microglia (gray arrowheads) of a female (a) and a male (b) embryo at embryonic day 12 (E12) compared to the fully ramified microglia (black arrow) of an adult (male) (c) immunostained with the QH1 antibody which also stains blood vessels (empty arrow). (d) Fluorescent photomicrographs illustrating the co-labeling of embryonic microglia of a male (QH1) with iNOS. (e) Fluorescent photomicrographs illustrating the co-labeling of embryonic microglia of a female immunostained with QH1 antibody and the RCA-1 lectin. VIII, third ventricle. Scale bars, 50 um [Color figure can be viewed at wileyonlinelibrary.com ]

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Experimental details: 9 FIGURE Effect of sex and minocycline on the intensity of inducible nitric oxide synthase (iNOS) immunofluorescence of individual microglia in the medial preoptic nucleus. The statistical boxes provide the results of separate two-way ANOVAs (performed on square-root transformed data) with sex and treatment as independent factors. Abbreviations: CTRL, control; Mino 50 or 500, minocycline 50 or 500 ug; N.S., non significant. The number of animals per group is written on each bar

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Experimental details: Figure 3 Prostaglandin E 2 (PGE 2 ) production and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in BV2 cells. Cell supernatant PGE 2 levels ( A ), protein expression of iNOS and COX-2 ( B ), and gene expression of iNOS and COX-2 relative to that of GAPDH ( C ) were determined using ELISA ( n = 6), Western blot ( n = 3), or real-time PCR ( n = 3) methods. Semiquantification of protein expression was performed with normalization to beta-actin ( D ). The data are expressed as the mean +- SD. # p < 0.05 and ## p < 0.01 compared with the vehicle-treated cells; * p < 0.05 and ** p < 0.01 compared with the LPS-treated BV2 cells.

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Experimental details: Fig. 8 CRH-c-Abl dependent mitochondrial fission requires NF-kB activity. A IHC for MAP2 (gray), VGlut1 (red) Shank2 (green) after different treatments (scale bar = 5 mum). White arrowheads indicate the co-localization between VGlut1 and Shank2. Quantification of excitatory synapses number (co-localization of Shank2/Vglut1/30mum of dendrites). Three different dendrites of three different neurons acquired from three different wells were analyzed for each condition in each independent experiment. B WB analysis of DRP1 S616 expression, blocking the NF-kappaB pathway with the nuclear translocation blocker JSH-23. C IHC for MAP2 (gray) and c-Abl T754 (red) in primary neurons after different experimental conditions using JSH-23 (scale bar = 30 mum): vehicle, vehicle + JSH 10 muM, CRH 100 nM for 0.5 h, CRH 100 nM + JSH 10 muM with relative c-Abl T754 intensity analysis. Nine different neurons acquired from three different wells were analyzed for each condition in each independent experiment. Representative western blot and relative quantification of: D S-nitrosylation levels of DRP1 and E of iNOS expression following CRH treatment; F iNOS expression levels in 5 Txt mice. Experiments were performed in N = 3 independent replicates at DIV14. Data are displayed as Mean +- SEM; one-way ANOVA and Bonferroni's post hoc comparison test were performed ( *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001).

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Experimental details: Figure 7 Colocalization of inflammatory molecules with neuronal and glial markers in CA3 region of 3xTg-AD mice. Double immunofluorescence staining for (A-C) TNF, (D-F) COX-2, and (G-I) iNOS with MAP2, GFAP, and IBA1 in the hippocampal CA3 region in 3xTg-AD mice. The results demonstrated that TNF-alpha, COX-2, and iNOS were detected in the cytoplasm of neurons (labeled with MAP2), astrocytes (labeled with GFAP), and microglial cells (labeled with IBA1), as indicated by the white arrowheads. TNF-alpha, COX-2 and iNOS (red); MAP2, GFAP, and IBA1 (green); and DAPI (blue) (nuclei). Bar = 50 mum.

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Experimental details: Figure 2 Assessment of neuro-inflammation trigged by bacterial conditioned media at two different concentrations. ( a ) Schematic diagram showing experimental timeline for neuro-inflammation assessment. ( b ) Morphological changes and immunostaining images against CD86, TREM-2 and iNOS markers of microglia cells treated with 10 ng/mL LPS, [1 BCM L :10 MM] and [1 BCM H :10 MM] in a single-culture system at day 4, scale bar 50 um. ( c - e ) Quantification of TREM2, CD86 and iNOS fluorescent intensity expressed by stained microglia cells. Significant differences among BCM L , BCM H and LPS treatment, compared with the control. ( f ) Analysis of the amount of nitric oxide released by microglia cells. ( g , h ) Quantification of chemokines and cytokines released in microglia-stimulated conditioned media indicated increased inflammatory response from microglia cells, compared with the control. All experiments were repeated three times independently and statistically analyzed by One-way ANOVA followed by Tukey HSD test (* p < 0.05, ** p < 0.01 and *** p < 0.001). All data were presented as mean +- standard deviation (SD).

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Experimental details: Figure 6 Inhibition of succinate dehydrogenase and LPS reducing microglial inflammatory responses. ( a ) Schematic showing experimental timeline for inhibition assay. ( b ) Immunostaining images against TREM-2, CD86 and iNOS markers of microglia cells. ( c - e ) Quantification of TREM2, CD86 and iNOS fluorescent intensity expressed by stained microglia cells. All experiments were repeated three times independently and statistically analyzed by One-way ANOVA followed by Tukey HSD test (* p < 0.05, ** p < 0.01). All data were presented as mean +- standard deviation (SD).

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Experimental details: Figure 7 Effects of indomethacin alone and with oral pre-treatments of famotidine or A. anthelmintica (200, 100 mg/kg) on ( A ) gastric inhibitor of nuclear factor kappa-B kinase subunit beta (IKKB), ( B ) gastric nuclear factor KB (NF-kappaB), ( C ) gastric TNF-alpha, and ( D ) gastric IL-6 in rats. Statistical analyses were carried out by one-way ANOVA followed by Tukey's post hoc test, mean +- SEM. * Significantly different from control group at p < 0.05. @ Significantly different from indomethacin alone group at p < 0.05.

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Experimental details: Fig. 4 Glioma tissues from GD3S-KO mice expressed higher levels of iNOS, and primary cultured glioma cells from WT mice expressed higher levels of pro-inflammatory cytokines Fig. 4a: Glioma tissues generated in WT and GD3S-KO mice were resected at 1 and 2 weeks after DF-1 cell injection, and those sections were stained by anti-CD68 antibody and anti-iNOS antibody. Alexa488-labeled goat-anti-mouse IgG antibody and Alexa568-labeled anti-rabbit IgG antibody were used as secondary antibodies, respectively. Three samples each were examined, and representative data are shown. Bars, 50 mum. Fig. 4b: Total RNA was extracted from WT and GD3S-KO mouse-derived primary cultured glioma cells. Gene expression levels were analyzed by RT-qPCR. Two samples each were analyzed 3 times. Student's t-test was performed for evaluation of the results. **p

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Experimental details: Figure 3 Macrophage infiltration is decreased in serum amyloid A double-knockout (SAADKO) mice. Representative immunohistochemical images of the distal colon of wild-type and SAADKO mice stained with antibodies for F4/80, inducible nitric oxide synthase (iNOS), and CD206, and hematoxylin as counterstain, along with quantified scoring results for each marker. *** P < .001 and **** P < .0001.

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Experimental details: Fig. 9 Immunofluorescent staining of the tissue surrounding the implants after subcutaneous implantation for 8 weeks. Fig. 9

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Experimental details: Figure 1 Impact of IL-1beta on nitric oxide (NO) release and NO synthase (iNOS) expression of human chondrocytes. ( a ) NO release after stimulation with Interleukin (IL)-1beta at different concentrations. Statistical differences vs. unstimulated cells, repeated-measures ANOVA ( n = 7). ( b ) NO release after stimulation with IL-1beta (0.1 ng/mL) and L-NIL at different concentrations. Statistical differences vs. IL-1beta-stimulated cells, repeated-measures ANOVA ( n = 7). ( c ) NO release after stimulation with 0.1 ng/mL IL-1beta and 50 uM forskolin. Statistical differences *** vs. unstimulated cells or ### vs. IL-1beta-stimulated cells, Wilcoxon signed-rank test and Bonferroni adjustment ( n = 22). ( d ) NO release after simultaneous stimulation of 0.1 ng/mL IL-1beta with 50 uM forskolin ( n = 22), 50 uM 8-Bromo-cAMP ( n = 10), or 10 ug/mL PGE 2 ( n = 9) normalized to IL-1beta-stimulated cells. Statistical differences vs. IL-1beta-stimulated cells, Wilcoxon signed-rank test. ( e ) Impact of activation or inhibition of AMPK on IL-1beta-induced NO release. Statistic: Repeated-measures ANOVA with Post Hoc test and Bonferroni adjustment ( n = 9). ( f ) NO release after stimulation with 50 uM forskolin, 10 uM L-NIL, 10 uM A769662, or 10 uM dorsomorphin dihydrochloride (DoMo). Statistical differences vs. unstimulated cells, Wilcoxon signed-rank test ( n = 5). ( g ) Representative Western blot of iNOS expression after stimulation with/without 0.1 ng/mL IL-1beta, 50 uM forskolin, 10

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Experimental details: Presence of immune regulatory cells in lesions formed by HPV16 E6/E7-expressing keratinocytes. A: Top panel: FOXP3 + cell density on day 8 and 12 lesions formed by MOKE6E7 and MOKE6E B cells. Bottom panels: CD4 and FOXP3 immunohistochemistry (IHC) analyses on the day 12 lesions. CD4 and FOXP3 IHC analyses: Red arrows mark positive brown signals. FOXP3/CD4 double IHC: Nuclear blue color represents FOXP3 signals, and membranous red color represents CD4 signals. Almost all FOXP3 + cells detected in both lesions were also CD4 + , which represented regulatory T cells, but not vice versa. B: Top panel: CD68 + cell density on day 8 and 12 lesions. Bottom panels: CD68, arginase 1 (ARG1), and nitric oxide synthase (NOS) 2 IHC analyses on the day 12 lesions. Red arrows mark the CD68 signals. Macrophages present in the lesions are polarized to M2. C: CD4, FOXP3, and CD68 IHC analyses on day 28 and 42 lesions of MOKE6E7 B . Images are representative of the studied mice in the group. Data represent means +- SD ( A and B ). N = 6. * P

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Experimental details: Figures (A-C) show the expression and differentiation of microglia before epilepsy modeling. (A) Western blot showing protein expression of Iba1 protein. Data are expressed as the mean +- SEM (n = 5 per group). (B) Western blot showing protein expression of Arg-1 protein. Data are expressed as the mean +- SEM (n = 5 per group). (C) Western blot showing protein expression of iNOS protein. Data are expressed as the mean +- SEM (n = 5 per group). Figures (D-I) show the expression and differentiation of microglia 24 hours after epilepsy modeling. (D) Western blot showing protein expression of Iba1 protein. Data are expressed as the mean +- SEM (n = 5 per group). (E) Iba1 positive cells (green; Alexa Fluor-488 staining) were observed with fluorescence microscope. White arrows indicate Iba1 positive cells. Scale bars: 100 mum for 200x, 50 mum for 400x. (F) Western blot showing protein expression of Arg-1 protein. Data are expressed as the mean +- SEM (n = 5 per group). (G) Western blot showing protein expression of CD68 protein. Data are expressed as the mean +- SEM (n = 5 per group). (H) Western blot showing protein expression of iNOS protein. Data are expressed as the mean +- SEM (n = 5 per group). (I) Western blot showing protein expression of CD86 protein. Data are expressed as the mean +- SEM (n = 5 per group). ns, no significance. *P < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA (and nonparametric or mixed) followed by methods of multiple comparisons.

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Experimental details: Figure 3. EZH2 silencing palliates neuroinflammation in depression rats by regulating microglia polarization. (a) Positive rate of iba-1 analyzed by immunofluorescence. (b) mRNA levels of iNOS, CD16, Arg-1 and CD206 detected by RT-qPCR. (c) Protein levels of iNOS and Arg-1 determined by Western blot analysis. (d) Contents of IL-1beta, TNF-alpha, IL-4 and IL-10 measured by ELISA. N = 6. The results in panels A, C and D were presented as mean +- standard deviation. One-way ANOVA was used to analyze the data in panels A, B and D. Two-way ANOVA was used to analyze the data in panel C. Tukey's multiple comparisons test was applied for post hoc test. ** p < 0.01.

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Experimental details: Figure 4. EZH2 silencing triggers M2 polarization of LPS-induced . (a) Positive rate of iba-1 in LPS-induced microglia analyzed by immunofluorescence. (b) mRNA levels of EZH2 detected by RT-qPCR. (c) mRNA levels of iNOS, CD16, Arg-1 and CD206 detected by RT-qPCR. (d) Protein levels of EZH2, iNOS and Arg-1 determined by Western blot analysis. (e) Contents of IL-1beta, TNF-alpha, IL-4 and IL-10 measured by ELISA. The experiment was performed 3 times. The results were presented as mean +- standard deviation. One-way ANOVA was used to analyze the data in panel B. Two-way ANOVA was used to analyze the data in panels C, D and E. Tukey's multiple comparisons test was applied for post hoc test. * p < 0.05, ** p < 0.01.

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Experimental details: Figure 6. Inhibition of miR-29b-3p expression mitigates the role of EZH2 silencing in LPS-induced microglia polarization. miR-29b-3p inhibitor was transfected into the microglia in the sh-EZH2 group, with the sh-NC as the control. (a) miR-29b-3p expression assessed by RT-qPCR. (b) Transcription levels of MMP2 detected by RT-qPCR. (c) mRNA levels of iNOS, CD16, Arg-1 and CD206 detected by RT-qPCR. (d) Protein levels of iNOS and Arg-1 determined by Western blot analysis. E, contents of IL-1beta, TNF-alpha, IL-4 and IL-10 measured by ELISA. The experiment was performed 3 times. The results were presented as mean +- standard deviation. One-way ANOVA was used to analyze the data in panels A and B. Two-way ANOVA was used to analyze the data in panels C, D and E. Tukey's multiple comparisons test was applied for post hoc test. * p < 0.05, ** p < 0.01.

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Experimental details: Histological analysis of the therapeutic effect of 4-OI@Zn-NH-pyr on acute arthritis in TMJ. (A) Representative images of H&E, S&F, TRAP, and immunostaining of iNOS and Nrf2 on day 7 after administration of various treatment groups (10 days after immunization) (n = 3 independent animals). (B) Representative images of H&E, S&F, TRAP, and immunostaining of iNOS and Nrf2 on day 14 after administration of various treatment groups (17 days after immunization) (n = 3 independent animals). (C) Quantitative analysis of the synovitis score. (D) Quantitative analysis of the OARSI score. (E) Quantitative analysis of the number of TRAP-positive OCs. (F) Quantitative analysis of the percentage of iNOS-positive cells. (G) Quantitative analysis of the percentage of Nrf2-positive cells. Data are presented as the mean +-SD (n = 3 independent animals). Statistical significance was determined by one-way ANOVA with Tukey's multiple comparison test. Black arrow, TRAP-positive OCs; SM, synovial macrophages; AC, articular cavity. * P < 0.05; ** P < 0.01.

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Experimental details: Figure 5. Immunofluorescence images of iNOS and CD206 in tissues adjacent to various samples ( a ). H&E staining of the tissues adjacent to various samples ( b ). The blue dotted lines indicate the fibrous layers

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Experimental details: Macrophages were actively involved in inflammatory bone remodeling and M1 macrophages were the major activated macrophage subtype in OA bone remodeling areas. A (i) H&E, TRAP, and IHC staining of macrophage specific markers (CD68: macrophage pan marker; iNOS: M1 macrophage marker; CD206: M2 macrophage marker; positive cells were labeled with red arrows) on normal and OA bone sections (a and f: scale bars represented was 200 mum in H&E staining; b and g: scale bars represented was 20 mum in TRAP staining; c, d, e, h, i, and j: scale bars represented was 20 mum in IHC staining); A(ii): The box plot graphs demonstrated the positive cells per field of view. Data was shown as the mean +- SD (* p < 0.05, t-test); B IF double staining of macrophage markers on normal and OA bone sections (CD68 and CD11b: macrophage pan markers; CD86: M1 macrophage marker, CD206: M2 macrophage marker; scale bars represented 20 mum); C IF double staining of M1 macrophage marker and osteocyte marker on OA bone sections (CD86: M1 macrophage marker; E11: early osteocyte marker; DMP1: mature osteocyte marker; scale bars represented 20 mum)

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Experimental details: Figure 4 MSC-Ex shifted the macrophage phenotype from M1 to M2 in the colon of mice with chronic colitis. The relative expressions of the macrophage phenotype-associated genes were evaluated by qRT-PCR: (A,B) MCP-1 and CXCL9 (M1 marker), (C,D) Arg-1 and IL-10 (M2 marker). (E) Representative photomicrographs of CD68 (macrophage marker), iNOS (M1 marker), and Arg-1 (M2 marker), immunohistochemically labeled in the colonic tissues of colitis mice. The photomicrographs were acquired at 100x magnification. (F) The protein levels of intracellular iNOS and Arg-1 were evaluated by western blotting. Data are represented as the means +- s.d. ( n >= 3 ). * P < 0.05, **P < 0.005, ***P < 0.001 .

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Experimental details: Figure 4 The inflammation-related protein expressions. ( A ) NF-kappaB, iNOS, and COX-2 expressions in RAW 264.7 cells were pretreated with P. emblica samples (0.25-2 mg/mL) for 1 h, and then were stimulated with LPS (5 mug/mL) for 6 h. ( B1 ) Protein quantification of NF-kappaB ( B2 ) iNOS ( B3 ) COX-2 in western blotting. beta-Actin was viewed as an internal control. * p < 0.05, ** p < 0.01.

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Experimental details: Figure 7 Genic and protein expression in thoracic aortas from wild-type and CD mice. ( A ) qPCR mRNA expression of Limk1 , a gene contained in the Williams-Beuren syndrome critical region, and Adra1a (alpha1 A -adrenoceptors). Results are the mean +- SEM from wild-type (n = 4) and CD (n = 5) mice. * P < 0.05; *** P < 0.01 by Multiple t-test with Sidak-Bonferroni. ( B ) Western blot analysis for nNOS protein expression. Bar graphs show the results of densitometric analyses from pooled data for nNOS, iNOS, and eNOS protein expression. The molecular weight (kDa) of the protein is shown on the right side of the blot. Total protein was used as a loading control. Results are the mean +- SEM from wild-type (n = 5) and CD (n = 5) mice. * P < 0.05 by Mann-Whitney U test. ( C ) Representative photomicrographs and quantification of immunofluorescence (red) for nNOS, iNOS, and eNOS in confocal microscopic aortic sections. Scale bar, 20 mum. Results are the mean +- SEM from wild-type (n = 8) and CD (n = 8) mice. * P < 0.05 by Student's t-test.

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Experimental details: Figure 1 iNOS immunolabeling of amoeboid microglia in retinas of quail embryos at 8 and 9 days of incubation. Confocal micrographs of QH1 (green) and anti-iNOS (red) double-immunolabeled amoeboid microglial cells in whole-mounted retinas (WM) and retinal cross-sections (CS) of quail embryos at 8 (E8, A , B ) and 9 days of incubation (E9, C , D ). Cell nuclei in retinal cross sections are stained with Hoechst (blue). All QH1-positive amoeboid microglial cells in E8 and E9 embryo retinas are located in the nerve fiber layer (NFL) and are also immunolabeled with the anti-iNOS antibody. Anti-iNOS labeling of microglial cells is similar to QH1 labeling but with some differences. Thus, QH1 labels the entire microglial cell profile, including lamellipodia, whereas anti-iNOS labeling is exclusively cytoplasmic in distinct zones of the soma and cell processes. OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar, 50 um for A and C; 60 um for B and D.

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Experimental details: Figure 1 Narrow-leafed lupin (NLL) gamma-conglutin decreases the mRNA expression and protein levels of TNFalpha, IL-1beta, and iNOS on lipopolysaccharide (LPS)-induced inflammation pancreatic cells. PANC-1 cells were incubated for 24 h with LPS alone or gamma-conglutin + LPS. ( A ) The bar graph shows mRNA levels determined by real-time RT-qPCR of TNFalpha, iNOS, and IL1beta. ( B ) The bar graph shows protein levels determined by immunoblotting of TNFalpha, iNOS, and IL1beta. Average value from triplicate experiments of each biomarker were relativized to the average value of their housekeeping actin protein in control samples. Then, average values from challenge experiments (calculated in the same way than controls) are relativized to these from their respective control values previously calculated. Data represent mean +- SD from three independent experiments. C: Untreated control culture cells; LPS: LPS-treated culture cells; LPS + gamma: LPS + gamma-conglutin challenge. p < 0.05 represents statistically significant differences associated with each figure. p * < 0.05 LPS versus C; p ** < 0.05 LPS + gamma-conglutin versus LPS. Challenges were made with LPS and/or gamma-conglutin protein at 1 mug/mL and 25 mug, respectively.

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Experimental details: Figure 2 MSC-Ex restores the PPARalpha activity in HaCaT cells. ( A ) HaCaT cells were treated as described in Fig. 1 and western blot analyses were performed using primary antibodies for PPARalpha, NF-kappaB-65, p-NF-kappaB-p65 Ser536 , IkappaB, p-IkappaB Ser32/36 and iNOS. beta-Actin was used as the loading control. ( B ) The protein levels of NF-kappaB were determined by ELISA. Columns represent the means of three independent experiments; bars indicate standard deviations.). * P < 0 . 05 , **P < 0 . 005 , ***P < 0 . 001 .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Juanbi-Tang (JBT) suppressed inflammatory cells and production of CCL2, CXCR3, and CCR6. (A) Immunofluorescence staining with F4/80/iNOS marking macrophages of collagen-induced arthritis (CIA) mice. (B) Immunofluorescence staining with F4/80/iNOS marking macrophages of tumor necrosis factor transgenic (TNF-Tg) mice and WT littermates. (C) Immunofluorescence staining with CD3 labeling T cells of CIA mice. (D) Immunofluorescence staining with CD3 labeling T cells of TNF-Tg mice and WT littermates. (E) Immunohistochemical staining with CCL2, CXCR3, and CCR6 of CIA mice. (F) Immunohistochemical staining with CCL2, CXCR3, and CCR6 of TNF-Tg mice and WT littermates.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 PGRN deficiency aggravates neuroinflammation and apoptosis after SCI. a At day 7 after SCI, total protein was extracted from injured spinal cord of both WT and PGRN-deficient mice, and iNOS, p-p65, p65, and GAPDH were detected by Western blotting. b Quantification of iNOS/GAPDH in ( a ). n = 3 per group. c Quantification of p-p65/p65 in ( a ). n = 3 per group. d Immunofluorescence of iNOS and CD68 in both WT and PGRN-deficient mice at day 7 after SCI. Images were taken at the anterior horn of gray matter. Arrows indicate co-localization of iNOS and CD68. e At day 7 after SCI, total protein was extracted from injured spinal cord of both WT and PGRN-deficient mice, and Bax, Bcl-2, and GAPDH were detected by Western blotting. f Quantification of Bax/GAPDH in ( e ). n = 3 per group. g Quantification of Bcl-2/GAPDH in ( e ). n = 3 per group. h At day 7 after SCI, apoptotic neurons in the ventral horn were detected by TUNEL assays. i Quantification of apoptotic neuron numbers in ( h ). n = 3 for each group. * p < 0.05, ** p < 0.01 WT-SCI vs Grn -/- -SCI groups. ns, not significant

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 6 Local delivery of Atsttrin improves neuroinflammation and apoptosis in PGRN-deficient mice after SCI. a BMS was used to evaluate the therapeutic effect of hydrogel/Atsttrin in both WT and PGRN-deficient mice. Asterisk indicates p < 0.05 between Grn -/- -SCI-gel/PBS and Grn -/- -SCI-gel/Atsttrin groups. b At day 7 after SCI, total protein was extracted from injured spinal cord of PGRN-deficient mice and iNOS, p-p65, p65, and GAPDH were detected by Western blotting. Quantification of iNOS/GAPDH ( c ) and p-p65/p65 ( d ) in ( b ). n = 3 for each group. e At day 7 after SCI, total protein was extracted from injured spinal cords of PGRN-deficient mice and Bax, Bcl-2, and GAPDH were detected by Western blotting. Quantification of Bax/GAPDH ( f ) and Bcl-2/GAPDH ( g ) in ( e ). n = 3 for each group. * p < 0.05, ** p < 0.01 for indicated groups, ns, not significant