mouse anti rabbit igg human lung tlr8 novus bio  (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: The FASEB Journal

Article Title: Propofol directly binds to and inhibits TLR7

doi: 10.1096/fj.202200312r

Figure Lengend Snippet: FIGURE 4 The impact of propofol on TLR7 dimer formation and MyD88 interaction. (A) TLR7 protein was subjected to crosslinking experiment using glutaraldehyde with or without R837 and/or propofol (PPF). R837-induced dimer formation. Dimer and monomer formations were quantitated by densimetry. (B) HEK-TLR7 cells were stimulated with R837 in the presence or absence of PPF 50 μM. Then, cell lysates were immunoprecipitated (IP) with TLR7, followed by Western bloting (WB) of MyD88 (left). Densimetry analysis is shown. Immunoprecipitated TLR7 was also probed and shown on the right. ***p < .001 using one-way ANOVA with Bonferroni post hoc analysis.

Article Snippet: Following centrifugation, supernatants were subjected to immunoprecipitation using rabbit anti- human TLR7 antibody (Cell Signaling technology; Danvers, MA) and Dynabeads Protein A immunoprecipitation kit (Thermofischer Scientific).

Techniques: Immunoprecipitation, Western Blot

Journal: The FASEB Journal

Article Title: Propofol directly binds to and inhibits TLR7

doi: 10.1096/fj.202200312r

Figure Lengend Snippet: FIGURE 3 Propofol-binding sites on TLR7. PDB5ZSF was used for the analysis. (A) R837-binding sites on TLR7 dimer (TLR7/TLR7*). R837 binds to the two sites (Site 1-1 and site 1-2). (B) Coverage map for TLR7 mass spectrometry analysis of photolabeled samples with propofol. Sequenced residues are shown in orange. Adducted residues are shown in red. (C) Propofol-binding sites on TLR7 dimer are shown. Based on the photolabeling and rigid docking experiments, five propofol-binding sites were identified. (D) R837-binding sites and propofol-binding sites are shown on TLR7 dimer together. R837 is shown in red arrow. Propofol is shown in blue circle.

Article Snippet: Following centrifugation, supernatants were subjected to immunoprecipitation using rabbit anti- human TLR7 antibody (Cell Signaling technology; Danvers, MA) and Dynabeads Protein A immunoprecipitation kit (Thermofischer Scientific).

Techniques: Binding Assay, Mass Spectrometry

Journal: The FASEB Journal

Article Title: Propofol directly binds to and inhibits TLR7

doi: 10.1096/fj.202200312r

Figure Lengend Snippet: FIGURE 5 The role of hydroxyl group in propofol in TLR7 binding. Fropofol is a propofol derivative where the hydroxyl (-OH) group was replaced with fluoride. (a) The effect of propofol (PPF) and fropofol (FPL) on TLR7 activation was examined. HEK-TLR7 cells were stimulated with R837 (10 mg/ml) in the presence of PPF and FPL (10 μM, 100 μM). Data were shown as mean ± SD of eight replicates. Two-way ANOVA was done. ***p < .001. (B) Coverage map for TLR7 mass spectrometry analysis of photolabeled samples with propofol. Sequenced residues are shown in orange. Adducted residues are shown in red. (C) Fropofol-binding sites on TLR7 dimer are shown. Based on the photolabeling and rigid docking experiments, two fropofol-binding sites were identified. (D) R837-binding sites and fropofol-binding sites are shown on TLR7 dimer together. R837 is shown in red arrow. Fropofol is shown in purple circle.

Article Snippet: Following centrifugation, supernatants were subjected to immunoprecipitation using rabbit anti- human TLR7 antibody (Cell Signaling technology; Danvers, MA) and Dynabeads Protein A immunoprecipitation kit (Thermofischer Scientific).

Techniques: Binding Assay, Activation Assay, Mass Spectrometry

Journal: iScience

Article Title: Neuregulin-1/ErbB4 signaling modulates Plasmodium falciparum HRP2-induced damage to brain cortical organoids

doi: 10.1016/j.isci.2022.104407

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-human TLR7 (clone D7) , Cell Signaling , Cat #5632T RRID: AB_10692895.

Techniques: Functional Assay, Recombinant, Plasmid Preparation, CCK-8 Assay, Bicinchoninic Acid Protein Assay, Isolation, Gene Expression, Western Blot, Software, Microscopy