PA3-030A

antibody from Invitrogen Antibodies

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

Provider product page for PA3-030A

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [78]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [4]
Immunohistochemistry [3]
Other assay [33]

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Product number: PA3-030A - Provider product page
Provider: Invitrogen Antibodies
Product name: iNOS Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA3-030A detects inducible nitric oxide synthase (iNOS) from human and mouse tissues. This antibody does not detect endothelial NOS (eNOS) or brain NOS (bNOS). PA3-030A has been successfully used in Western blot and immunohistochemistry (paraffin and frozen) procedures. By Western blot, this antibody detects an ~130 kDa protein representing iNOS in induced RAW 264.7 cell extracts. The PA3-030A immunogen is a synthetic peptide corresponding to residues C K(1131) K G S A L E E P K A T R L(1144) of mouse macrophage NOS. PA3-030A can be used with blocking peptide PEP-008. Antibodies to this protein (and modification) were previously sold as part of a Thermo Scientific Cellomics High Content Screening Kit. This replacement antibody is now recommended for researchers who need an antibody for high content cell based assays. It has been thoroughly tested and validated for cellular immunofluorescence (IF) applications. Further optimization including the selection of the most appropriate fluorescent Dylight conjugated secondary antibody may have to be performed for your high content assay.
Reactivity: Human, Mouse
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Concentration: Conc. Not Determined
Storage: -20° C, Avoid Freeze/Thaw Cycles

NOS2 protein structure - PA3-030A shown in red.

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Experimental details: Immunofluorescent analysis of iNOS using Anti-iNOS Polyclonal Antibody (Product # PA3-030A) shows staining in NIH-3T3 Cells. iNOS staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing iNOS (Product # PA3-030A) at a dilution of 1:200 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552, Goat Anti-Rabbit). Images were taken at 60X magnification.

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Experimental details: Immunofluorescent analysis of iNOS using Anti-iNOS Polyclonal Antibody (Product # PA3-030A) shows staining in Hela Cells. iNOS staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing iNOS (Product # PA3-030A) at a dilution of 1:200 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552, Goat Anti-Rabbit). Images were taken at 60X magnification.

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Experimental details: Immunofluorescent analysis of iNOS using Anti-iNOS Polyclonal Antibody (Product # PA3-030A) shows staining in Neuro-2a Cells. iNOS staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing iNOS (Product # PA3-030A) at a dilution of 1:200 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552, Goat Anti-Rabbit). Images were taken at 60X magnification.

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Experimental details: Immunofluorescence analysis of iNOS was performed using 70% confluent log phase A549 cells treated with a combination of TNF-α (5 ng/mL), IFNγ (50 U/mL) and IL-1β (50 U/mL) for 18 h. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with iNOS Rabbit Polyclonal Antibody (Product # PA3-030A) at 1:250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic and nuclear localization. Panel e shows the untreated cells with no signal. Panel f represents the no primary control. The images were captured at 60X magnification.

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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized Mouse liver 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 # PA3-030A) 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 biopsies of deparaffinized Mouse 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 1:100 with a rabbit polyclonal antibody recognizing iNOS (Product # PA3-030A) 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 biopsies of deparaffinized Mouse lymph node 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 # PA3-030A) 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: Fig. 3 Protein expression of GFAP, Notch-1, NICD, HES-1, TNF-alpha, IL-1beta and iNOS in TNC1 following different treatments. a Expression levels of GFAP, Notch-1, NICD, and HES-1 were significantly increased after treatment with CM + L when compared with the control in CM; likewise, the expression of TNF-alpha, IL-1beta and iNOS was significantly increased. b In CM + SL treated TNC1, the expression levels of the above markers were further elevated as compared with that of CM + L treated cells. Bar graphs represent expression changes of the respective markers. Significant differences in protein levels are expressed as * # P < 0.05. The values represent the mean +- SD in triplicate. # CM + L vs CM, *CM + SL vs CM + L

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Experimental details: Fig. 5 TRPV4 activation by LPS triggers NO production in human bronchial EC. a Immunoblotting of 16HBE cells lysate probed for NOS isoforms. b Left, average intracellular Ca 2+ responses of human bronchial epithelial cells to 20 mug ml -1 LPS and 10 nM GSK1016790A. The black, blue, and red solid traces correspond to data means of cells responding to LPS and GSK1016790A, GSK1016790A alone or to none of the stimuli, respectively. The thin dashed traces represent the means +- standard errors. Right, stack bar graph showing the percentages of cells responding to LPS and/or GSK1016790A (color-coded as the graph on the left; n = 116). c Immunoblotting of 16HBE cells lysate incubated in LPS (20 mug ml -1 ) or GSK1016790A (10 nM). Where indicated, cells were previously incubated with vehicle (Veh, 0.8% DMSO) or the TRPV4 inhibitor HC067047 (+HC, 10 muM). d Average intracellular Ca 2+ signals (black solid traces) and normalized fluorescence of the NO-sensitive dye DAF-FM (F/F 0 , red solid traces) recorded in 16HBE cells ( n = 52). The thin dashed traces represent the means +- standard errors. LPS, 20 ug ml -1 ; HC067047, 10 uM; GSK1016790A, 10 nM. e Average changes in intracellular Ca 2+ (left panel) and NO production (right panel) induced by LPS. The dark cyan bars correspond to cells pre-incubated with the TRPV4 inhibitor HC067047 (10 uM). n > 74 per bar; ** P < 0.01, two-tailed t -test. f Confocal microscopy images of 16HBE cells immunostained for iNOS. The arrow head points to an

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Experimental details: Figure 1. UK021 cancerous (CA) lung tissue is less mitotic, has less nitric oxide (NO)-producing potential, and is more necrotic than UK049 CA lung tissues. UK021 and UK049 CA and noncancerous (NC) lung tissues were sampled in the operating room and immediately preserved in formalin for immunohistochemical analysis for PCNA (mitotic index marker), RIP-1 (necrosis marker), and iNOS (inducible nitric oxide synthase, marker for M1-like macrophages [ Ohri et al. 2009 , 2011 ]) as green fluorescence, CD68 (general macrophage marker) as red fluorescence, and DAPI (nuclear marker) as blue fluorescence. ( A , C ) An example frame of the PCNA, RIP-1 (60x magnification), and iNOS + CD68 fluorescence images (60x magnification), as well as their normalized image intensity averaged over three to seven image frames for UK021 tissues. The fluorescence intensity of PCNA/RIP-1 and iNOS was respectively normalized to that of DAPI and CD68. ( B,D ) The corresponding image frames and normalized image intensity for UK049 tissues are shown. The UK021 CA lung tissue displayed a relatively lower mitotic index, higher necrotic state, and lower iNOS to CD68 ratio than the UK049 CA tissue. For both patients, the CA lung tissues were more mitotic and necrotic with a higher iNOS/CD68 ratio than the NC lung tissues proximal and distal to the CA tissues.

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Experimental details: Figure 7. beta-Glucan-enhanced iNOS expression in UK021 but not UK049 CA lung tissue slices. WGP treatment, tissue preservation, and immunohistochemical analysis are as in Figure 6 . As in Figure 1 , the green fluorescence (iNOS) was normalized to the red fluorescence (CD68) to reflect WGP-induced increase in iNOS expression relative to CD68 expression for UK021 ( A ) but not for UK049 ( B ) CA lung tissue slices.

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Experimental details: Fig. 2 Immunohistochemical staining demonstrates the intensity of iNOS expression in the cytoplasm of papillary carcinoma cases. ( A ) score 0; ( B ) score 1; ( C ) score 2; ( D ) score 3 (x400).

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Experimental details: Immunolocalization of M1-like macrophages in the decidual vessels with acute atherosis. Multiplex immunofluorescence staining showing nuclear staining (4',6-diamidino-2-phenylindole, DAPI, blue), smooth muscle actin (SMA, green), cytokeratin-7 (CK7, yellow), CD68 (red), CD80 (pink), interleukin (IL)-12A (purple), and inducible nitric oxide synthase (iNOS, cyan) in the decidua basalis with acute atherosis. Representative images showing (A) the immunohistochemistry and fluorescence views and (B) magnification of an M1-like macrophage in the vessel wall of a non-transformed decidual vessel with acute atherosis. Representative images showing (C) the immunohistochemistry and fluorescence views and (D) magnification of an M1-like macrophage near a transformed decidual vessel with acute atherosis. Phenoptics was performed to generate separate and merged immunofluorescence images (B & D) and to convert fluorescence images to the immunohistochemistry view (A & C). Black arrows in the immunohistochemistry view and dotted boxes in the fluorescence view indicate an M1-like macrophage. Images are representative of three experiments per group. Images were taken at 200x magnification, and a close-up of an M1-like macrophage is shown. Scale bars = 50 um (original image) or 5 um (close-up image)

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Experimental details: Figure 7 Representative images for immunohistological staining (brown) for iNOS in the hindlimbs of CIA mice that were untreated ( A and B ), or treated with Dex ( C and D ), low ND-ODA ( E and F ), high ND-ODA ( G and H ) or ND-ODA-Dex ( I and J ). N = 2-3 hindlimbs on 1-3 mice. Images magnified at x4 (left column) and x20 (right column). The boxed sections represent the area that has been magnified. All scale bars are 200 mum

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Experimental details: Figure 4 Multiplex IHC analysis of PTGS2 co-localization with macrophage markers CD68, iNOS, Arg1, MRC1/CD206, and CD163: ( a ) two examples of co-localization signals in the M1 series (CD68-iNOS-PTGS2), pseudocolors have been obtained overlaying the black&white mask of each marker on the first slide of the series; ( b ) two examples of co-localization signals in the M2 series (Arg1-MRC1-CD163-PTGS2); ( c ) left, mean Pearson's coefficients of PTGS2 and CD68 co-localization; right: a simplified representation of the mean overlay between PTGS2 and each marker using Manders' overlap coefficients. Slices indicate the extent of PTGS2 signal in the positive areas of each marker (complete analysis is shown in Supplementary Figure S2 ).

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Experimental details: Fig. 2 Miconazole reduces neuroinflammation in LPS-induced mice hippocampus. The immunostaining of GFAP ( a ), Iba-1 ( b ), iNOS ( c ), and COX-2 ( d ) proteins in the hippocampus was performed in 10-um-thick sections of mice brains with specific primary antibodies and biotinylated secondary antibodies. In the upper-right values mean the dyed density in the hippocampus area (DG, CA1, CA3). Quantifications of the areas positively labeled with GFAP, Iba-1, iNOS, or COX-2 were performed using the ImageJ. Quantification results ( n = 3 mice per group) were analyzed using two-way ANOVA, followed by Bonferroni posttests. Data are expressed as the mean +- SEM (two-way ANOVA for repeated measures followed by post-hoc Bonferroni, # p < 0.05 ## p < 0.01, ### p < 0.0001 vs. Con, $ p < 0.05, $$ p < 0.01, $$$ p < 0.0001 vs. MCZ, * p < 0.05, ** p < 0.01, *** p < 0.0001 vs. MCZ + LPS). Representative images of immunohistochemistry staining with respective antibodies are shown here. Full immunohistochemistry results are included in Supplementary Fig. 4 .

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Experimental details: Figure 1 Knockdown of CISD2 expression augmented tendency for M1 microglial polarization and enhanced proinflammatory response in non-stressed EOC microglial cells. The conditions were set as follows: (i) scrambled RNA-transfected control cells; (ii) siCID2-transfected cells. (A) Confirmation of knockdown efficiency of CISD2 mRNA and protein (B) expression in microglia. (C) Expression of TNF-alpha mRNA, (D) IL-1beta mRNA, (E) iNOS mRNA, and (F) COX2 mRNA under all conditions. (G) iNOS protein expression in siCID2-transfected EOC microglial cells. The upper panel demonstrates the immunoblot results of iNOS at 135 kDa, and beta-actin (45 kDa) serves as an internal control; the lower panel indicates the mean (+-SEM) of iNOS/beta-actin band intensity in ratio with the control group. Vertical bars indicate the mean +- (SEM) of mRNA or protein expression ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001 indicate statistically significant difference when compared to scrambled control cells.

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Experimental details: Figure 3 CMS reduced oxidative stress, inflammation, and apoptosis in DR rats. STZ-treated rats were treated with DMSO, 10 mg/kg CMS, 50 mg/kg CMS, and 100 mg/kg CMS. n = 15 per treatment. ( A ) Expression pattern of iNOS as determined by RT-qPCR in rat retinal tissues, normalized to beta-actin. ( B ) Representative Western blots of iNOS protein and its quantitation in rat retinal tissues, normalized to beta-actin. ( C ) Expression of NO measured by nitrite test in rat retinal tissues. ( D - F ) Expression patterns of IL-6 ( D ), TNF-alpha ( E ), and CRP ( F ) measured by ELISA in the cell supernatant. ( G , H ) Representative images of apoptotic cells (x 400) (scale bar = 25 mum) ( G ) and cell apoptosis in rat retinal tissues ( H ) by TUNEL. ( I ) Representative Western blots of cleaved caspase-3 protein and its quantitation in rat retinal tissues, normalized to beta-actin. ( J , K ) Representative Western blots of Cyt-C protein and its quantitation in the cytoplasm and mitochondria, normalized to beta-actin. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the sham-operated rats, and # p < 0.05, ## p < 0.01, ### p < 0.001, compared to the rats injected with STZ and treated with DMSO. The results were measurement data, which were expressed as mean +- standard deviation. Comparisons between multiple groups were analyzed by one-way ANOVA with Tukey''s post hoc test (n = 15). iNOS, inducible nitric oxide synthase; CMS, coumestrol, STZ, streptozotocin; DMSO, dimethyl sulfoxi

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Experimental details: Figure 6 CMS suppressed HG-induced oxidative stress and inflammation in hRMECs through activating SIRT1. hRMECs were treated with sh-NC, sh-SIRT1, sh-SIRT1 + DMSO and sh-SIRT1 + CMS, respectively. ( A ) Expression pattern of iNOS as determined by RT-qPCR in hRMECs, normalized to beta-actin. ( B ) Representative Western blots of iNOS protein and its quantitation in hRMECs, normalized to beta-actin. ( C ), Expression pattern of NO as determined by nitrite test in hRMECs. ( D - F ), Expression patterns of IL-6 ( D ), TNF-alpha ( E ), and CRP ( F ) as measured by ELISA in the cell supernatant. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to sh-NC-treated cells, and # p < 0.05, ## p < 0.01, ### p < 0.001, compared to cells stimulated with sh-SIRT1 and treated with DMSO. The results were measurement data and expressed as mean +- standard deviation. Comparisons between multiple groups were analyzed by one-way ANOVA with Tukey''s post hoc test. The cell experiments were repeated three times independently. NC, negative control; CMS, coumestrol, HG, high glucose; hRMECs, human retinal microvascular endothelial cells; SIRT1, sirtuin 1; DMSO, dimethyl sulfoxide; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; IL-6, interleukin-6; TNF-alpha, tumor necrosis factor alpha; CRP, C-reactive protein; ELISA, Enzyme linked immunosorbent assay; ANOVA, analysis of variance.

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Experimental details: Fig. 1 Treatment with Agac lysate in combination with tensile strain increased NADPH oxidase 4 (NOX-4) and nitric oxide synthase-2 (NOS-2). Gene ( a ) and protein expression of NOX-4 ( b ) and NOS-2 ( c ) after treatment with Agac lysate and tensile strain (cropped blots from Additional file 1 : Figure S4); n = 6; AU arbitrary units. Statistics: ordinary ANOVA with Holm-Sidak's post hoc (NOX-4 mRNA and protein) or Welch-corrected ANOVA with Games-Howell post-hoc tests (NOS-2); * p

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Experimental details: Fig. 4 Impact of Agac lysate in combination with compressive strain on development of reactive oxygen species (ROS) and expression of nitric oxide synthase-2 (NOS-2). Gene ( a ) and protein expression of NADPH oxidase-4 ( b NOX-4), development of ROS ( c ) and NOS-2 ( d ) after treatment with Agac lysate and compressive strain (cropped blots from Additional file 1 : Figure S5); n >= 6; AU arbitrary units. Statistics: ordinary ANOVA with Holm-Sidak's post hoc or Welch-corrected ANOVA with Games-Howell post-hoc tests (NOS-2); * p

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Experimental details: Fig. 4 The polarization of macrophages to M2 phenotype is related to the elongated cell shape. A Fluorescence micrographs of Raw 264.7 macrophages immune-stained for arginase-1 (green), iNOS (red), and nuclear counterstain (blue) on cell plate (control), collagen and HA-collagen nanofibrous films. Scale bars: 15 mum. B Representative Western blot of arginase-1, iNOS, and tubulin of control, collagen and HA-collagen nanofibrous films and quantification of average across three separate experiments. Quantified TNF-alpha C and IL-10 D secretion from macrophages cultured on different nanofibrous scaffolds or culture plates using ELISA assay. n = 3, ** p < 0.01, *** p < 0.001

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Experimental details: Figure 5 Chewing behavior during psychological stress improved oxidative stress in the tumor. ( A ) The expression level of iNOS protein in the tumor. ( B ) The expression level of 4HNE protein in the tumor. ( C ) The expression level of SOD2 protein in the tumor (** p < 0.01 vs. control group, ## p < 0.01 vs. stress group, n = 9/group). All data are expressed as mean +- SEM.

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Experimental details: Figure 5 Hepatic inflammatory factor expressions in the mice fed with the liquid Lieber-DeCarli ethanol diet. ( A ) Immunoblotting photograph of the ( B ) TNF-alpha, ( C ) IL-1beta, ( D ) IL-6, ( E ) NF-kappaB, ( F ) iNOS, ( G ) COX-2, ( H ) ERK1/2, ( I ) p-ERK/ERK1/2 ratio, ( J ) p38, and ( K ) p-p38/p38 ratio. Two groups of the mice were fed a normal diet (NOR group) or a liquid Lieber-DeCarli ethanol diet (EtOH group) without the administration of test materials. The other ethanol-induced liver injury mice were administered silymarin (200 mg/kg bw/day), a low dose of monascin (0.615 mg/kg bw/day; MS-L group), a high dose of monascin (3.075 mg/kg bw/day; MS-H group), ankaflavin (0.3075 mg/kg bw/day; AK-L group), or a high dose of ankaflavin (1.5375 mg/kg bw day; AK-H group). Data are presented as mean +- SD ( n = 8). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. EtOH group. Abbreviations: TNF-alpha, tumor Necrosis Factor-alpha; IL-1beta, Interleukin 1 beta; IL-6, Interleukin 6; NF-kappaB, nuclear factor kappa-light-chain-enhancer of activated B; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; and ERK1/2, extracellular signal-regulated kinase 1/2.

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Experimental details: Fig. 4 Combined re-expression of ASS1 and ASL suppresses growth in vivo. A Combined doxycycline-induced ectopic expression of ASS1+ ASL in 786-O cells. HSP90 is used as a loading control. B 786-O subcutaneous xenograft tumor growth in nude mice, where cells have doxycycline induced expression of ASS1 and ASL or empty vector ( n = 6). C Immunohistochemistry staining and quantification for phosphorylated histone H3 as a marker for proliferation in xenograft tumors. Scale bars are at 100 mum. D Immunohistochemistry staining and quantification for cleaved caspase 3 as a marker for apoptosis in xenograft tumors. Scale bars are at 100 mum. ** p < 0.01. E Immunohistochemistry staining and quantification for p21 as a marker for proliferation in xenograft tumors. Scale bars are at 100 mum. *** p < 0.001. F Nitric oxide metabolite levels in matched ccRCC tumors and normal kidney as measured using gas-phase chemiluminescence ( n = 20). G Nitric oxide synthase activity measured by radiolabeled citrulline generation, plotted as a fold change between matched normal and ccRCC tumor tissues ( n = 20). H Representative immunohistochemistry images with quantification for NOS1,2,3 protein expression in matched tumor-normal pairs (Biomax OD-CT-UrKid03-002) ( n = 31). Scale bars represent 200 mum. **** p < 0.001, paired t -test

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Experimental details: Western blot and gene expression analysis of LPS-induced hippocampal injury, resulting inflammatory signaling pathways. Mice were sacrificed and brain tissues were removed after the final LPS injection. The hippocampus was isolated from brain tissue and prepared either protein lysates ( n = 6-9 to each group) or mRNA ( n = 4-6 to each group) samples were prepared for further analysis. qPCR analysis was used to determine the mRNA expression levels of pro-inflammatory cytokines including ( A ) Tnfa, ( B ) Il1b, and ( C ) Il6. Western blot analysis of ( D ) NLRP3, iNOS, p-p65, p-65, IkappaBalpha, TLR4, and actinin were performed ( n = 4 to each group). The data were expressed as mean +- SEM. ## p < 0.01 and ### p < 0.001 for Con vs. LPS, * p < 0.05, and ** p < 0.01 for LPS vs. drug-administrated groups.

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Experimental details: Spinal cord injury (SCI) downregulates Cisd2 and enhances inflammatory response in the injured spinal cord of mice subjected to spinal cord hemisection. The secretion of various inflammatory mediators in the cerebrospinal fluid (CSF) of rats with SCI. ( A ) Experimental groups and the schedule of treatment with anti-CISD2 antibody in a mouse model of SCI. ( B ) The spinal cord levels of Cisd2 mRNA in the 1, 24, and 48 h post-SCI groups were significantly decreased compared with those in the sham operation group. ( C ) Under the same conditions as those in ( B ), the spinal cord levels of Tnfa mRNA were significantly increased. For B and C, vertical bars indicate mean +- standard error of mean (SEM) ( n = 6 for each group). * p < 0.05, ** p < 0.01, and *** p < 0.001 (pair-wise multiple comparisons between groups were performed using the Newman-Keuls test). The spinal cord levels of the Cisd2 protein were decreased ( D ) and those of Nos2 were increased ( E ) in the 24 h post-SCI group compared with those in the sham operation group. For D and E, the upper panel indicates representative immunoblot of the protein expression of Cisd2 (15 kDa) ( D ) and Nos2 (135 kDa); ( E ) Gapdh (35 kDa) was used as an internal control. For ( D ) and ( E ), vertical bars indicate mean +- SEM of protein levels ( n = 3 for each group). * p < 0.05, sham operation group vs. SCI groups (independent two-sample t -tests). ( F ) Detection of inflammatory mediators in the CSF using the cytokine antibody

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Experimental details: Fig. 2 miR-17 inhibits cartilage destruction by targeting multiple pathological catabolic factors. a Luciferase activity of 293 T cells co-transfected with dual-luciferase reporter constructs containing wild-type or mutated 3'-UTRs, as well as negative control (mimic NC) or miR-17 mimic. The data presented are mean percentage changes over mimic NC +- s.e.m. n = 6 biologically independent samples (NC + wt UTR, miR-17+wt UTR); n = 4 biologically independent samples (NC + mut UTR, miR-17 + mut UTR). b Mouse articular chondrocytes were transfected with miR-17 mimic (50 nM) or mimic NC and treated with or without IL-1beta (5 ng/mL) for 24 h. The protein levels of catabolic and anabolic factors were determined by western blot followed by densitometry analysis. Blots are representative of three independent experiments. c , d qRT-PCR analysis of catabolic genes in knee cartilage ( c ), and representative images of IHC staining and quantification of MMP13 + , ADAMTS5 + and NOS2 + cells in joint sections ( d ) of mice subjected to sham or DMM surgery and intra-articular injections of agomir-NC or agomir-17 (1.5 nmol) for 4 weeks, beginning at 4 weeks after surgery. n = 5 biologically independent samples per group in c . For MMP13 and ADAMTS5 staining, n = 4 mice (sham); n = 5 mice (DMM, DMM + agomir-17). For NOS2 staining, n = 4 mice per group. e , f qRT-PCR analysis of catabolic genes ( e ) and representative images of MMP13, ADAMTS5, and NOS2 immunostaining, and quantification of pos

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Experimental details: Endogenous miR-17 was upregulated by GDF-5 to suppress cartilage destruction in DMM mice. a , b FISH for miR-17 and safranin O/fast green staining of joint sections ( a ), and quantification of miR-17-positive cells and OARSI scores ( b ). Veh (PBS) or GDF-5 (100 ng per injection) was injected weekly for 4 weeks, beginning at 4 weeks after sham or DMM surgery. n = 4 mice per group for cell counting. For OARSI grade, n = 4 mice (sham); n = 6 mice (DMM); n = 5 mice (DMM + GDF-5). c - e qRT-PCR analysis of catabolic genes ( c ); safranin O/fast green staining and IHC staining for MMP13, ADAMTS5 and NOS2 ( d ); OARSI scores; and quantification of cells positive for immunostaining ( e ) from knee joints. A combination of GDF-5 (0 or 100 ng) and 3 nmol of the antagomir (antagomir-17 or antagomir-NC) was injected intra-articularly. The injections were performed weekly for 4 weeks, beginning at 4 weeks after sham or DMM surgery. n = 4 biologically independent samples (sham + antagomir-NC) and n = 5 biologically independent samples for other groups in ( c ). For OARSI scores, n = 4 mice (sham); n = 7 mice (DMM); n = 9 mice (GDF-5 + antagomir-NC, GDF-5 + antagomir-17). For MMP13 staining, n = 5 mice (antagomir-17); n = 4 mice in other groups. For NOS2 and ADAMTS5 staining, n = 4 mice per group. All scale bars, 100 mum. Data were presented as boxplots or mean +- s.e.m. Boxplots: center line, median; box limits, 25th to 75th percentiles; whiskers, min to max. * P < 0.05, ** P < 0.01, and

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Cartilage degeneration was developed in miR-17~92 cKO mice. a , b Safranin O/fast green staining and H&E staining of knee joint cartilage ( a ), OARSI grade ( n = 6 mice in control and cKO 8wk; n = 9 mice in cKO; n = 8 mice in cKO + agomir-17), cartilage thickness ( n = 6 mice per group) and cell counts of superficial chondrocytes ( n = 5 mice in control; n = 6 mice for other groups) in femoral condylar cartilage ( b ) from miR-17~92 fl/fl (control), miR-17~92 cKO and agomir-17-injected cKO mice at 4 or 8 weeks after tamoxifen injection, were presented. The arrows in ( a ) indicated cartilage degeneration (left panels) and superficial chondrocytes (right panels). The triangles in ( a ) indicated splitting in cartilage matrix. c , d IHC staining of MMP13, ADAMTS5 and NOS2 ( c ), and qRT-PCR analysis of catabolic factors ( d ) in cartilage of control and miR-17~92 cKO mice at 4 weeks after tamoxifen injection. n = 4 mice per group in ( c ) and n = 3 mice per group in ( d ). Data were presented as boxplots (center line, median; box limits, 25 to 75th percentiles; whiskers, min to max), or the mean +- s.e.m. values. * P < 0.05, and *** P < 0.001. Mann-Whitney U test (two sided) for OARSI grade; one-way ANOVA with Bonferroni's test for cartilage thickness and cell number; two-sided Student's t test for qRT-PCR. All scale bars, 100 mum. Exact P values in ( d ): 0.2236 ( Mmp3 ), 0.7143 ( Mmp13 ),0.0513 ( Adamts5 ), 0.8648 ( Nos2 ). Source data are provided as a Source Data file .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 5 Inducing effect on macrophage polarization of cells RAW264 cultured with leaching solutions from PLA, 1% and 5% BG-PLA. (A) . RT-PCR; (B) Immunofluorescence.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Obesity-induced adipose inflammation is attenuated by lactate depletion in adipocytes. Wildtype (WT) and adipocyte-specific Ldha knockout (AKO) mice were fed with standard chow (STC) or high fat diet (HFD) for 4 months before epididymal white adipose tissue (eWAT) was isolated for analysis. a HE staining of eWAT; scale bar, 100 mum. b Immunofluorescence staining of iNOS and F4/80 in eWAT; scale bar, 100 mum. c - f SVF in eWAT was subjected to flowcytometry analysis for macrophage subtypes. STC-WT: n = 5 ( c - f ); STC-AKO: n = 4 ( c-f ); HFD-WT: n = 12 ( d ) or n = 11( e ) or n = 10 ( f ); HFD-AKO: n = 7 ( c - f ) biologically independent animals. g - j mRNA expression of g Il-1beta (STC-WT: n = 5; STC-AKO: n = 4; HFD-WT: n = 6; HFD-AKO: n = 6 biologically independent animals), h Cd11c (STC-WT: n = 4; STC-AKO: n = 4; HFD-WT: n = 7; HFD-AKO: n = 6 biologically independent animals), i Tnf (STC-WT: n = 4; STC-AKO: n = 4; HFD-WT: n = 9; HFD-AKO: n = 6 biologically independent animals) and j Ccl12 (STC-WT: n = 4; STC-AKO: n = 4; HFD-WT: n = 8; HFD-AKO: n = 7 biologically independent samples) in eWAT. k - l Serum levels of k IL-1beta and l MCP1. n = 4 biologically independent animals. Data represent mean +- SEM; Significance was calculated by two-way ANOVA with post hoc Bonferroni correction ( d-i ).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Lactate potentiates IL-1beta expression in mouse and human inflammatory macrophage. a - d Conditioned medium (CM) of epididymal white adipose tissue (eWAT) was collected from wildtype (WT) and adipocyte-specific Ldha knockout (AKO) mice. Bone marrow derived macrophages (BMDM) were cocultured in CM for 24 hr. a Illustration of the co-culture experiment. b , c BMDM mRNA expression of b Il-1beta , n = 4 biologically independent animals, and c Tnf , STC-WT: n = 4; STC-AKO: n = 3; HFD-WT: n = 3; HFD-AKO: n = 4 biologically independent animals. d Il-1beta mRNA in BMDMs cocultured in CM from WT and AKO eWAT. One group of BMDM was cocultured in CM from obese eWAT with AZD3965 (100 nM). n = 4 biologically independent samples. e - i Unelicited or inflammatory BMDMs were treated with 20 mM lactate or 20 mM NaCl as control in vitro. e - f Lactate content in cell lysate was measured after BMDM was cultured with medium containing 20 mM C3- 13 C lactate for 24 hr. e Lactate containing zero, 1, 2 and 3 13 Carbons (m + 0, m + 1, m + 2, m + 3) in cell lysate. n = 3 biologically independent samples. f Relative enrichment of endogenous and m + 1 13 C lactate in cell lysate. g , h mRNA level of g Il-1beta and h Tnf . n = 4 biologically independent samples. i Concentration of IL-1beta in BMDM medium. n = 4 biologically independent samples. j mRNA level of iNOS . n = 4 biologically independent samples. k Western blotting of iNOS. l - n Human CD14 + monocytes were differentiated to macrophages and t

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Human adipose lactate level is positively correlated with adipose inflammation and insulin resistance independent of BMI. a - e Correlation between lactate levels in human omental adipose tissues with a BMI, b fasting insulin, c fasting glucose, d HOMA-IR and e HOMA-beta index. n = 65 subjects. f - m 7 omental fat samples were randomly selected from low and high lactate groups (Low_Lac vs. High_Lac). f Inflammatory cytokines mRNA level in adipose. n = 7 individuals. g HE staining of omental fat; scale bar, 200 mum. Arrows point at the crown like structures (CLSs). h CLS numbers in omental fat. n = 7 individuals. i , j Immunofluorescence staining of iNOS and HIF-1alpha and human macrophage marker CD14; scale bar, 40 mum. k , l RNASeq of omental fat ( n = 7 individuals). k GO enrichment of upregulated pathways in High_Lac group. l Heatmap showing differentially expressed genes in GO:0002274 and GO:0002253. m Transcription factor (TF) enrichment in upregulated genes in High_Lac group. Significantly enriched TFs ( p < 0.05) were highlighted in blue. HIF-1alpha was shown in the enlarged region. n Illustration of the study hypothesis. Data represent mean +- SEM; Statistics were performed using spearman correlation analysis ( a - e ), two-way ANOVA with post hoc Bonferroni correction ( f ) or two-tailed student's t test ( h ).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Continuous infusion of IIK-7 attenuated PSNT-induced activation of HMGB-1, pERK1/2, iNOS, STAT3 and caspase-3 in vivo. Representative immunoblots are HMGB-1, P44/42 MAPK, iNOS, STAT 3 and casp-3 from the left dorsal quadrant portion of the lumbar spinal cord lysate of sham surgery or PSNT rats continuously infused with either vehicle or IIK-7 for 7 days using an osmotic pump. Abbreviations: IIK-7, N-Butanoyl 2-(9-methoxy-6H-iso-indolo[2,1-a]indol-11-yl)-ethan-amine; P44/42 MAPK, P44/42 mitogen-activated protein kinase; iNOS, Inducible nitric oxide synthase; STAT3, Signal transducer and activator of transcription 3; CASP-3, caspase3; HMGB-1, High mobility group box 1.