rabbit anti human tlr7 polyclonal antibody  (Novus Biologicals)

Journal: Nature Communications

Article Title: TLR7 promotes smoke-induced experimental lung damage through the activity of mast cell tryptase

doi: 10.1038/s41467-023-42913-z

Figure Lengend Snippet: a TLR7 mRNA levels in airway epithelial brushings from non-smokers (NS), healthy smokers without COPD (Smoker) and COPD patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I (mild) or II (moderate) disease ( n = 12 NS; n = 12 Smokers; n = 15 mild or moderate COPD). b TLR7 mRNA levels in lung parenchyma cores from NS and COPD patients with GOLD stage IV (severe) disease ( n = 16 NS; n = 48 severe COPD). Differential gene expression analysis was performed using published microarray datasets (GEO accession numbers GSE5058 and GSE27597) and the numbers in panels a and b represent the false discovery rate (FDR), whereby *denotes FDR of COPD vs . NS; and # denotes FDR of COPD vs . Smoker. The data are presented as box and whiskers with min to max showing all points. c Correlation analysis of anti-Smith antibody levels in serum and forced expiratory volume in 1 second (FEV 1 ) of mild-to-moderate COPD patients ( n = 40). d Human lung sections stained with tryptase, TLR7 and DAPI by immunofluorescence, and e TLR7 + mast cells were enumerated in sections from NS controls ( n = 4), smoker ( n = 6) and COPD patients ( n = 11). The numbers of TLR7 + mast cells correlated with f FEV 1 % predicted, g pack years of cigarettes, and h low attenuation areas less than a threshold of −950 Hounsfield units (%LAA950) in NS, smoker, and COPD patients. i Induction of experimental COPD where wild-type (WT) BALB/c mice (female, 6–8 weeks old) were exposed to nose-only inhalation of cigarette smoke (CS) for up to 12 weeks, controls received normal air. j Tlr7 mRNA levels in whole lungs of WT mice exposed to normal air or CS after 4, 6, 8, and 12 weeks ( n = 6 mice per group). Tlr7 mRNA levels in blunt-dissected k airways and l lung parenchyma after 8 weeks of CS exposure ( n = 6 mice per group). Wild-type (WT) BALB/c mice ( n = 6) were exposed to CS for 8 weeks to induce experimental COPD, controls were exposed to normal air. m TLR7 protein was assessed in mouse lungs by immunoblot, and n quantitated by densitometry analysis of fold change normalised to β-actin ( n = 6 mice per group). o Representative micrographs ( n = 3 mice per group) of TLR7 immunostaining in small airways (top) and lung parenchyma (bottom) of WT mice exposed to normal air (left) or CS (right) for 8 weeks. Scale bars, 50 µm. WT BALB/c mice were exposed to 8 weeks of CS, control mice breathed normal air. p Total TLR7 + cells, q mMCP4 + TLR7 + mast cells and r F4/80 + TLR7 + macrophages enumerated in whole lung sections ( n = 6 mice per group). All data are presented as means ± s.e.m. and are representative of two independent experiments. For panel c , f – h , correlation analyzes were performed using Spearman’s rank correlation coefficient test. For panel e , compared to NS or smokers using one-way ANOVA with Bonferroni’s multiple comparison test. The rest of the panels compared COPD to normal air-exposed controls using a two-tailed Mann–Whitney test. Source data are provided as a Source Data file.

Article Snippet: Then, using the ULTRA Staining system (Ventana Medical Systems), sections were washed with deionized water and incubated (37˚C, 30 min) with primary rabbit polyclonal anti-human TLR7 antibody (dilution 1:100, Proteintech, code 17232-1-AP, Supplementary Table ).

Techniques: Expressing, Microarray, Staining, Immunofluorescence, Western Blot, Immunostaining, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: TLR7 promotes smoke-induced experimental lung damage through the activity of mast cell tryptase

doi: 10.1038/s41467-023-42913-z

Figure Lengend Snippet: Wild-type (WT) BALB/c mice and Tlr7 −/− mice (female, 6–8 weeks old) were exposed to cigarette smoke (CS) or normal air for 8 weeks. a Quantification of destructive index ( n = 6 per group). b Representative micrographs (left) of hematoxylin and eosin-stained lung sections from WT (top panels) and Tlr7 −/− (bottom panels) mice exposed to normal air (left panels) or CS (right panels). Scale bars, 200 µm. Quantification of mean linear intercept (right, n = 6 per group). c Representative micrographs (left) of TUNEL-stained lung sections from WT (top panels) and Tlr7 −/− (bottom panels) mice exposed to normal air (left panels) or CS (right panels). Arrows indicate TUNEL + cells. Scale bars, 20 µm. Quantification of apoptotic cells (right, n = 5 per group). d Quantification of small airway epithelial cell area per µm of basement membrane (BM) perimeter and e nuclei numbers per 100 µm of BM perimeter of normal air- or CS-exposed WT and Tlr7 −/− mice (4 small airways per mouse, n = 6 per group). f Mouse lung sections were stained with Sirius red and fast green. Scale bar=50 um. g Quantification of collagen around the small airways of air- or CS-exposed WT and Tlr7 −/− mice (4 small airways per mouse, n = 6 per group). h Lung sections were stained with fibronectin by immunohistochemistry. Scale bar=50 um and i quantification of fibronectin around the small airways of air- or CS-exposed WT and Tlr7 −/− mice (4 small airways per mouse, n = 6 per group). j Transpulmonary resistance of normal air- or CS-exposed WT and Tlr7 −/− BALB/c mice ( n = 6 per group). All data are presented as means ± s.e.m. and are representative of two independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison test. ns, not significant. Source data are provided as a Source Data file.

Article Snippet: Then, using the ULTRA Staining system (Ventana Medical Systems), sections were washed with deionized water and incubated (37˚C, 30 min) with primary rabbit polyclonal anti-human TLR7 antibody (dilution 1:100, Proteintech, code 17232-1-AP, Supplementary Table ).

Techniques: Staining, TUNEL Assay, Membrane, Immunohistochemistry, Comparison

Journal: Nature Communications

Article Title: TLR7 promotes smoke-induced experimental lung damage through the activity of mast cell tryptase

doi: 10.1038/s41467-023-42913-z

Figure Lengend Snippet: a Wild-type (WT) BALB/c mice (female, 6–8 weeks old) were administered low-dose imiquimod (50 μg in 50 μl sterile saline), intranasally (i.n.) 5 times per week, for 8 weeks. Controls received sterile saline. b Quantification of destructive index ( n = 6 mice per group) of saline- or imiquimod-administered WT mice. c Quantification of mean linear intercept ( n = 6 mice per group) and representative micrographs (right) of hematoxylin and eosin (H&E)-stained lung sections from saline (top panel)- or imiquimod (bottom panel)-administered WT mice. Scale bars, 200 µm. d Quantification of apoptotic cells ( n = 6 mice per group) and representative micrographs (right) of TUNEL-stained lung sections from saline (top panel)- or imiquimod (bottom panel)-administered WT mice. Arrows indicate TUNEL + cells. Scale bars, 20 µm. e Transpulmonary resistance of saline- or imiquimod-administered WT mice ( n = 6 mice per group). WT BALB/c mice (female, 6–8 weeks old, n = 8) were challenged with high-dose imiquimod (100 μg in 50 μl sterile saline) intranasally, 5 times per week, for 2 weeks. Controls were challenged with sterile saline. f Total leukocytes, g macrophages, and h lymphocytes in bronchoalveolar lavage fluid (BALF, n = 6 mice per group). mRNA expression of i Cxcl1 , j Tnf , k Infar1 were assess in lungs by qPCR ( n = 6 mice per group). l Lungs were stained with H&E (scale bar = 50 μm) and m alveolar diameter was assessed ( n = 6 mice per group). n Lung function, in terms of transpulmonary resistance was assessed using the flexiVent system ( n = 6 mice per group). WT BALB/c mice were administered 5×10 5 bone-marrow-derived mast cells intranasally from either WT or Tlr7 −/− mice. o Neutrophils and p mast cells were counted in BALF 3 days after receiving mast cells ( n = 5 mice per group). q WT BALB/c mice were exposed to normal air or CS for 8 weeks and some groups were administered imiquimod (50 μg in 50 μl sterile saline), i.n. 5 times per week, between Week 6 to 8 (for 2 weeks). Controls received sterile saline. r Quantification of the destructive index ( n = 6 mice per group) of saline- or imiquimod-administered WT mice exposed to normal air or CS for 8 weeks. s Quantification of mean linear intercept ( n = 6 mice per group) and representative micrographs (right) of H&E-stained lung sections from saline (top panel)- or imiquimod (bottom panel)-administered WT mice exposed to normal air (left panel) or CS (right panel) for 8 weeks. Scale bars, 200 µm. t Quantification of apoptotic cells ( n = 6 mice per group) and representative micrographs (right) of TUNEL-stained lung sections from saline (top panel)- or imiquimod (bottom panel)-administered WT mice exposed to normal air (left panel) or CS (right panel) for 8 weeks. Arrows indicate TUNEL + cells. Scale bars, 20 µm. u Transpulmonary resistance of saline- or imiquimod-administered WT mice exposed to normal air or CS for 8 weeks ( n = 8 mice per group). All data are presented as means ± s.e.m. and are representative of two independent experiments. For panels b – n statistical analysis was performed using two-tailed Mann–Whitney test. For the rest of the panels, statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison test. Source data are provided as a Source Data file.

Article Snippet: Then, using the ULTRA Staining system (Ventana Medical Systems), sections were washed with deionized water and incubated (37˚C, 30 min) with primary rabbit polyclonal anti-human TLR7 antibody (dilution 1:100, Proteintech, code 17232-1-AP, Supplementary Table ).

Techniques: Sterility, Saline, Staining, TUNEL Assay, Expressing, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Nature Communications

Article Title: TLR7 promotes smoke-induced experimental lung damage through the activity of mast cell tryptase

doi: 10.1038/s41467-023-42913-z

Figure Lengend Snippet: a Wild-type (WT) or TLR7-deficient ( Tlr7 −/− ) or MyD88-deficient ( Myd88 −/− ) BALB/c mice (female, 6–8 weeks old) were administered imiquimod (50 μg in 50 μl sterile saline), intranasally (i.n.) 5 times per week, for 2 weeks. Controls received sterile saline. b Quantification of destructive index ( n = 8 mice per group) of saline- or imiquimod-administered WT and Tlr7 −/− mice. c Quantification of mean linear intercept ( n = 8 mice per group) and representative micrographs (right) of hematoxylin and eosin (H&E)-stained lung sections from WT (top panels) and Tlr7 −/− (bottom panels) mice administered saline (left panels) or imiquimod (right panels). Scale bars, 200 µm. d Quantification of apoptotic cells ( n = 6 mice per group) and representative micrographs (right) of TUNEL-stained lung sections from WT (top panels) and Tlr7 −/− (bottom panels) mice administered saline (left panels) or imiquimod (right panels). Arrows indicate TUNEL + cells. Scale bars, 20 µm. e Transpulmonary resistance of saline- or imiquimod-administered WT and Tlr7 −/− mice ( n = 8 mice per group). f Quantification of destructive index ( n = 6 mice per group) of saline- or imiquimod-administered WT and Myd88 −/− mice. g Quantification of mean linear intercept ( n = 6 mice per group) and h representative micrographs of H&E-stained lung sections from WT (top panels) and Myd88 −/− (bottom panels) mice administered saline (left panels) or imiquimod (right panels). Scale bars, 200 µm. i Quantification of apoptotic cells ( n = 6 mice per group) and representative micrographs (right) of TUNEL-stained lung sections from WT (top panels) and Myd88 −/− (bottom panels) mice administered saline (left panels) or imiquimod (right panels). Arrows indicate TUNEL + cells. Scale bars, 20 µm. j Transpulmonary resistance of saline- or imiquimod-administered WT and Myd88 −/− mice ( n = 6 mice per group). All data are presented as means ± s.e.m. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison test. Source data are provided as a Source Data file.

Article Snippet: Then, using the ULTRA Staining system (Ventana Medical Systems), sections were washed with deionized water and incubated (37˚C, 30 min) with primary rabbit polyclonal anti-human TLR7 antibody (dilution 1:100, Proteintech, code 17232-1-AP, Supplementary Table ).

Techniques: Sterility, Saline, Staining, TUNEL Assay, Comparison

Journal: Nature Communications

Article Title: TLR7 promotes smoke-induced experimental lung damage through the activity of mast cell tryptase

doi: 10.1038/s41467-023-42913-z

Figure Lengend Snippet: a Quantification of mast cells in lung sections from wild type (WT) BALB/c mice (female, 6–8 weeks old, n = 6 mice per group) administered imiquimod or vehicle for 8 weeks. Quantification of mast cells in lung sections from b WT, TLR7- ( Tlr7 −/− ) or c MyD88-deficient ( Myd88 −/− ) BALB/c mice (female, 6–8 weeks old) administered imiquimod or vehicle for 2 weeks ( n = 8 mice per group). d Quantification of mast cells in lung sections from WT BALB/c mice exposed to normal air or CS for 8 weeks and administered imiquimod or vehicle from weeks 6–8 ( n = 6 mice per group). e WT mice were first administered cromolyn (50 mg/kg body weight) or vehicle (sterile water), and after 2 h, were administered imiquimod (50 μg) or vehicle. Cromolyn, imiquimod, and vehicle were delivered intranasally (i.n.) 5 times per week, for 2 weeks. f Quantification of the destructive index ( n = 8 mice per group) of vehicle- or imiquimod-administered mice with or without cromolyn treatment. g Quantification of mean linear intercept ( n = 8 mice per group) and representative micrographs (right) of hematoxylin and eosin (H&E)-stained lung sections from vehicle (top panels) and cromolyn (bottom panels) mice administered vehicle (left panels) or imiquimod (right panels). Scale bars, 200 µm. h Quantification of apoptotic cells ( n = 6 mice per group). i Transpulmonary resistance of saline- or imiquimod-administered mice with or without cromolyn treatment ( n = 8 mice per group). j WT or mouse mast cell protease-6-deficient ( mmcp6 −/− ) C57BL/6 mice were administered imiquimod (50 μg in 50 μl sterile saline), intranasally 5 times per week, for 2 weeks. Controls received sterile saline. k Quantification of destructive index ( n = 6 mice per group) of saline- or imiquimod-administered WT and mmcp6 −/− mice. l Quantification of mean linear intercept ( n = 6 mice per group) and representative micrographs (right) of H&E-stained lung sections from WT (top panels) and mmcp6 −/− (bottom panels) mice administered saline (left panels) or imiquimod (right panels). Scale bars, 200 µm. m Quantification of apoptotic cells ( n = 6 mice per group). n Transpulmonary resistance of saline- or imiquimod-administered WT and mmcp6 −/− mice ( n = 6 mice per group). All data are presented as means ± s.e.m. For panel a , statistical differences were determined by two-tailed Mann–Whitney test. For rest of panels, statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison test. Source data are provided as a Source Data file.

Article Snippet: Then, using the ULTRA Staining system (Ventana Medical Systems), sections were washed with deionized water and incubated (37˚C, 30 min) with primary rabbit polyclonal anti-human TLR7 antibody (dilution 1:100, Proteintech, code 17232-1-AP, Supplementary Table ).

Techniques: Sterility, Staining, Saline, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Nature Communications

Article Title: TLR7 promotes smoke-induced experimental lung damage through the activity of mast cell tryptase

doi: 10.1038/s41467-023-42913-z

Figure Lengend Snippet: a Representative micrographs (top panels, n = 3) and color deconvolution of isotype control (left panels) and TLR7 (right panels) immunostaining of HMC-1 human mast cells. Scale bars, 50 µm. b Representative micrographs (top panels, n = 3) and color deconvolution (bottom panels) of isotype control (left panel) and mast cell tryptase (right panels) immunostaining of HMC-1 cells incubated with media or imiquimod (5, 10 or 100 ng) for 1 h, Scale bars, 50 µm. Quantification of mast cell tryptase in cells (10 random fields per sample, n = 3 per group) normalized to c number of cells or d area of hematoxylin of HMC-1 cells incubated with media or imiquimod (5, 10 or 100 ng) for 1 h. e Quantification of mast cell tryptase activity ( n = 6 per group) in terms of p -nitroaniline levels in culture supernatants from HMC-1 cells incubated with media or imiquimod (5, 10, or 100 ng) for 1 h. Throughout, data are presented as means ± s.e.m. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison test. Source data are provided as a Source Data file.

Article Snippet: Then, using the ULTRA Staining system (Ventana Medical Systems), sections were washed with deionized water and incubated (37˚C, 30 min) with primary rabbit polyclonal anti-human TLR7 antibody (dilution 1:100, Proteintech, code 17232-1-AP, Supplementary Table ).

Techniques: Immunostaining, Incubation, Activity Assay, Comparison

Journal: Nature Communications

Article Title: TLR7 promotes smoke-induced experimental lung damage through the activity of mast cell tryptase

doi: 10.1038/s41467-023-42913-z

Figure Lengend Snippet: a Wild-type (WT) BALB/c mice (female, 6–8 weeks old) were exposed to normal air or CS for 8 weeks and treated with neutralizing anti-TLR7 monoclonal antibody or isotype control, intravenously (i.v.) once per week for 2 weeks, from weeks 6–8. b Quantification of destructive index ( n = 6 mice per group) in lungs of isotype- or anti-TLR7-treated WT mice exposed to normal air or CS for 8 weeks. c Quantification of mean linear intercept ( n = 6 mice per group) of isotype or anti-TLR7 -treated WT mice exposed to normal air or CS for 8 weeks. d Quantification of apoptotic cells ( n = 6 mice per group) in TUNEL-stained lung sections from isotype or anti-TLR7 treated WT mice exposed to normal air or CS for 8 weeks. Quantification of e small airway epithelial cell area per µm of basement membrane (BM) perimeter and f nuclei numbers per 100 µm of BM perimeter of isotype- or anti-TLR7-treated WT mice exposed to normal air or CS for 8 weeks (4 small airways per mouse, n = 6 mice per group). g Transpulmonary resistance of isotype- or anti-TLR7-treated WT mice exposed to normal air or CS for 8 weeks ( n = 6 mice per group). h Quantification of mast cells in lung sections from isotype- or anti-TLR7-treated WT mice exposed to normal air or CS for 8 weeks ( n = 6 mice per group). i Quantification of mast cells in lung sections from WT and Tlr7 −/− mice exposed to normal air or CS for 8 weeks ( n = 6 mice per group). j WT BALB/c mice (female, 6–8 weeks old) were exposed to normal air or CS for 12 weeks and treated with neutralizing anti-TLR7 monoclonal antibody or isotype control, i.v. once per week, from weeks 8–12 (for 4 weeks). Some mice had CS cessation others continued CS exposure after 8 weeks prior to anti-TLR7 treatment. k Quantification of destructive index, l mean linear intercept and m apoptotic cells ( n = 6 mice per group) in lungs of isotype- or anti-TLR7-treated WT mice exposed to normal air or CS with CS cessation or continued CS exposure from 8–12 weeks. Quantification of n small airway epithelial cell area per µm of basement membrane (BM) perimeter and o nuclei numbers per 100 µm of BM perimeter of isotype- or anti-TLR7-treated WT mice exposed to normal air or CS with CS cessation or continued CS exposure from 8–12 weeks (4 small airways per mouse, n = 6 mice per group). p Measurement of diffusing lung capacity for carbon monoxide (DL CO ) of isotype- or anti-TLR7-treated WT mice exposed to normal air or CS with CS cessation or continued CS exposure from 8–12 weeks ( n = 6 mice per group). q Quantification of mast cells in lung sections from isotype- or anti-TLR7-treated WT mice exposed to normal air or CS with CS cessation or continued CS exposure from 8–12 weeks ( n = 6 mice per group). r Schematic representation of proposed mechanisms of how TLR7 contributes to CS-induced apoptosis and emphysema-like alveolar enlargement in experimental COPD in a mast cell-specific tryptase-dependent manner. All data are presented as means ± s.e.m. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison test. Source data are provided as a Source Data file.

Article Snippet: Then, using the ULTRA Staining system (Ventana Medical Systems), sections were washed with deionized water and incubated (37˚C, 30 min) with primary rabbit polyclonal anti-human TLR7 antibody (dilution 1:100, Proteintech, code 17232-1-AP, Supplementary Table ).

Techniques: TUNEL Assay, Staining, Membrane, Comparison

Journal: Journal of Clinical Medicine

Article Title: Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

doi: 10.3390/jcm12051941

Figure Lengend Snippet: Antibodies and protocols for indirect immunofluorescence analysis.

Article Snippet: Polyclonal rabbit anti-human TLR7 (Novus Bio) , 0.05 M Tris HCl pH 8.5 in MW , 1:300 overnight at 4 °C.

Techniques: Immunofluorescence, Incubation

Journal: Journal of Clinical Medicine

Article Title: Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

doi: 10.3390/jcm12051941

Figure Lengend Snippet: TLR2, TLR4, TLR7, and TLR9 immunofluorescence analysis on paraffin human skin sections. Representative TLR2 ( A , B ), TLR4 ( C , D ), TLR7 ( E , F ), and TLR9 ( G , H ) immunostainings in normal human skin paraffin sections. ( A , C , E , G ): samples harvested at 24 h; ( B , D , F , H ): samples harvested at 48 h. ( A – H ): control samples. Nuclei are counterstained with DAPI. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; TLR7: Toll-like receptor 7; TLR9: Toll-like receptor 9; DAPI: 4′, 6-diamidino-2-phenylindoledihydrochloride. White dotted line indicates the basal membrane. White arrows indicate positive immunostaining—scale bars: 50 µm.

Article Snippet: Polyclonal rabbit anti-human TLR7 (Novus Bio) , 0.05 M Tris HCl pH 8.5 in MW , 1:300 overnight at 4 °C.

Techniques: Immunofluorescence, Control, Membrane, Immunostaining

Journal: Journal of Clinical Medicine

Article Title: Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

doi: 10.3390/jcm12051941

Figure Lengend Snippet: TLR2, TLR4, TLR7, and TLR9 immunofluorescence analysis on IL-4 and IL-13 incubated paraffin human skin sections. Representative TLR2 ( A – D ), TLR4 ( E – H ), TLR7 ( I – L ), and TLR9 ( M – P ) immunostainings in normal human skin paraffin sections. ( A , E , I , M , C , G , K , O ): samples harvested at 24 h; ( B , F , J , N , D , H , L , P ): samples harvested at 48 h. Nuclei are counterstained with DAPI. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; TLR7: Toll-like receptor 7; TLR9: Toll-like receptor 9; IL-4: interleukin 4; IL-13: interleukin 13; DAPI: 4′, 6-diamidino-2-phenylindoledihydrochloride. White dotted line indicates the basal membrane. White arrows indicate positive immunostaining—scale bars: 50 µm.

Article Snippet: Polyclonal rabbit anti-human TLR7 (Novus Bio) , 0.05 M Tris HCl pH 8.5 in MW , 1:300 overnight at 4 °C.

Techniques: Immunofluorescence, Incubation, Membrane, Immunostaining

Journal: Journal of Clinical Medicine

Article Title: Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

doi: 10.3390/jcm12051941

Figure Lengend Snippet: TLR2, TLR4, TLR7, and TLR9 immunofluorescence analysis on IL-22 and IL-23 incubated paraffin human skin sections. Representative TLR2 ( A – D ), TLR4 ( E – H ), TLR7 ( I – L ), and TLR9 ( M – P ) immunostainings in normal human skin paraffin sections. ( A , E , I , M , C , G , K , O ): samples harvested at 24 h; ( B , F , J , N , D , H , L , P ): samples harvested at 48 h. Nuclei are counterstained with DAPI. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; TLR7: Toll-like receptor 7; TLR9: Toll-like receptor 9; IL-22: interleukin 22; IL-23: interleukin 23; DAPI: 4′,6-diamidino-2-phenylindoledihydrochloride. White dotted line indicates the basal membrane. White arrows indicate positive immunostaining—scale bars: 50 µm.

Article Snippet: Polyclonal rabbit anti-human TLR7 (Novus Bio) , 0.05 M Tris HCl pH 8.5 in MW , 1:300 overnight at 4 °C.

Techniques: Immunofluorescence, Incubation, Membrane, Immunostaining

Journal: PLoS ONE

Article Title: Molecular Determinants of GS-9620-Dependent TLR7 Activation

doi: 10.1371/journal.pone.0146835

Figure Lengend Snippet: (a) Three dimensional molecular model of TLR7 endo-lysosomal domain (left) and magnified view of GS-9620 docked in TLR7 (right). (b,c) Fold increase in NF-κB-luciferase reporter activity upon GS-9620, resiquimod or DMSO control stimulation in Huh7 cells that were transfected with control vector, wild-type TLR7 or point mutants of TLR7. Four 2-fold dose titrations (left to right) were performed starting at 5μM for GS-9620, or 10 μM of resiquimod. Bar graphs show fold change of reporter activity relative to DMSO control, and error bars shown represent the mean of triplicate assay conditions and SEM. Representative data shown from three independent experiments with similar results. Area under the curve (AUC) calculations were performed to quantify reporter activity observed with titrated compound concentrations for each of the TLR7 variants. With GS-9620 stimulation the variants Y356S, F408A, D555A, L557D, and L557Y/T586G, and with R848 stimulation the variants Y356S, F408A, and D555A show a 4-fold or greater reduction in reporter activity compared to TLR7 WT, as assessed by AUC calculation. Therefore, these variants are viewed as significantly compromised in response to the respective compound.

Article Snippet: Rabbit anti-human TLR7 polyclonal Ab (PA1-20817, Fisher Scientific), mouse anti-V5-Tag monoclonal Ab (R960-25, Life Technologies), mouse anti-HA-Tag monoclonal Ab (H3663, Sigma-Aldrich), anti-GAPDH monoclonal Ab (G8795, Sigma-Aldrich), HRP-conjugated secondary antibodies, goat anti-mouse (A4416, Sigma-Aldrich) and goat anti-rabbit (A6154, Sigma-Aldrich) were used.

Techniques: Luciferase, Activity Assay, Control, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Molecular Determinants of GS-9620-Dependent TLR7 Activation

doi: 10.1371/journal.pone.0146835

Figure Lengend Snippet: Fold increase in NF-kB-luciferase reporter activity upon GS-9620 stimulation in Huh7 cells that were transfected with TLR7 or SNPs rs179008 (Q11L), rs55907843 (V222D), or rs5743781 (A448V). Four 2-fold dose titration curves were performed starting at 5µM for GS-9620 (left to right). Bar graphs show fold change in reporter activity relative to DMSO, and error bars represent the mean of triplicate assay conditions and SEM. AUC calculations were performed as discussed for , above. By the same AUC criteria none of the three assessed TLR7 SNP variants elicited a significantly altered response, relative to TLR7 WT, after GS-9620 stimulation. Representative data are shown from three independent experiments with similar results.

Article Snippet: Rabbit anti-human TLR7 polyclonal Ab (PA1-20817, Fisher Scientific), mouse anti-V5-Tag monoclonal Ab (R960-25, Life Technologies), mouse anti-HA-Tag monoclonal Ab (H3663, Sigma-Aldrich), anti-GAPDH monoclonal Ab (G8795, Sigma-Aldrich), HRP-conjugated secondary antibodies, goat anti-mouse (A4416, Sigma-Aldrich) and goat anti-rabbit (A6154, Sigma-Aldrich) were used.

Techniques: Luciferase, Activity Assay, Transfection, Titration

Journal: PLoS ONE

Article Title: Molecular Determinants of GS-9620-Dependent TLR7 Activation

doi: 10.1371/journal.pone.0146835

Figure Lengend Snippet: Immunoblot analysis of whole cell lysates of Huh7 cells transfected with V5-tagged TLR7 (TLR7-V5) or HA-tagged TLR7 (TLR7-HA) or HA-tagged TLR9 (TLR9-HA) untreated or treated with increasing amounts of GS-9620 (0.1μM, 1μM and 5μM) for 1 hour before preparation of cell lysates and immunoprecipitation (IP) analysis. Lysates were immunoprecipitated with anti-V5 agarose (panel C,D) and immunoblot probed with anti-V5 mAb (panel A,C) or anti-HA mAb (panel B,D). Total cell lysates used in panels A and B were probed with anti-V5 mAb and anti-HA mAb respectively, to control for protein expression and loading.

Article Snippet: Rabbit anti-human TLR7 polyclonal Ab (PA1-20817, Fisher Scientific), mouse anti-V5-Tag monoclonal Ab (R960-25, Life Technologies), mouse anti-HA-Tag monoclonal Ab (H3663, Sigma-Aldrich), anti-GAPDH monoclonal Ab (G8795, Sigma-Aldrich), HRP-conjugated secondary antibodies, goat anti-mouse (A4416, Sigma-Aldrich) and goat anti-rabbit (A6154, Sigma-Aldrich) were used.

Techniques: Western Blot, Transfection, Immunoprecipitation, Control, Expressing