rabbit anti human tlr2  (Novus Biologicals)

Journal: Gut Microbes

Article Title: ADP-heptose attenuates Helicobacter pylori -induced dendritic cell activation

doi: 10.1080/19490976.2024.2402543

Figure Lengend Snippet: TLR2 mediated Helicobacter pylori uptake is essential for potent dendritic cell activation. (a) TLR2 mRNA expression upon stimulation with H. pylori wt or mutant (Δ) (MOI 5) at indicated time points. Mean±SD of three individual donors is shown. (b) TLR2 surface expression was monitored by flow cytometry 16 h post-infection (MOI 5) ( n = 4). (c,d) H. pylori strains were stained with eFluor670 proliferation dye prior to infection. One hour post infection with H. pylori wt or the ADP-heptose-deficient mutant (Δ) (MOI 5), DCs were subjected to immunofluorescence and stained for CD45, DAPI and TLR2. Internalization of bacteria as well as TLR2 localization was analyzed by confocal fluorescence microscopy. Orthogonal views of confocal z-stacks of one out of three representative donors are shown. Scale bar: 5 µm. (e) DCs were treated with a TLR2 neutralizing antibody 20 min prior to infection. After 1 h of infection with eFluor670 stained H. pylori (MOI 5), DCs were subjected to immunofluorescence and stained for CD45 and DAPI. Maximum intensity projections of confocal z-stacks of one representative out of three donors are shown. Scale bar: 5 µm. (f,g) DCs were treated with a TLR2 neutralizing antibody 20 min prior to infection with H. pylori (wt) or the ADP-heptose deficient mutant (Δ) at an MOI of 5. After 16 h CD40 expression (f) and IL-12p70 secretion (g) was monitored by flow cytometry and multiplex assay ( n = 6). For statistical analysis one-way ANOVA with a šídák’s post-hoc test was performed.

Article Snippet: Following primary antibodies were used: mouse anti-human CD45 (ab30470 Abcam, 1:200), rabbit anti-human TLR2 (NB100–56720 Novus Biologicals, 1:400).

Techniques: Activation Assay, Expressing, Mutagenesis, Flow Cytometry, Infection, Staining, Immunofluorescence, Bacteria, Fluorescence, Microscopy, Multiplex Assay

Journal: Brain, behavior, and immunity

Article Title: TLR2 immunotherapy suppresses neuroinflammation, tau spread, and memory loss in rTg4510 mice.

doi: 10.1016/j.bbi.2024.08.002

Figure Lengend Snippet: Fig. 1. TLR2 knockout reduces tau pathology and alleviates cognitive functions in rTg4510 mice. (A) Differential RNA expression of TLRs in AD brains compared to cognitively normal controls. Log2 fold changes are indicated. ACC, anterior cingulate cortex; CBE, cerebellum; DLPFC, dorsolateral prefrontal cortex; FP, frontal pole; IFG, inferior frontal gyrus; PCC, posterior cingulate cortex; PHG, parahippocampal gyrus; STG, superior temporal gyrus; TCX, temporal cortex. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (B-D) Tlr2 KO ameliorates cognitive impairment in rTg4510 mice. The 6-month-old mice (tTA/WT, tTA/Tlr2 KO, rTg4510/ WT, rTg4510/Tlr2 KO) were analyzed with the Y-maze test [B, two-way ANOVA with Tukey test, n (male, female) = 10 (7, 3); 10 (2, 8); 8 (6, 2); 11 (4, 7)], novel object recognition test [C, two-way ANOVA with Tukey test, n (male, female) = 8 (5, 3); 7 (2, 5); 6 (4, 2); 7 (2, 5)], and passive avoidance test [D, paired t-test, n (male, female) = 10 (7, 3); 10 (2, 8); 9 (6, 3); 11 (4, 7)]. (E-G) Tlr2 KO reduces tau pathology and microglial activation in rTg4510 mice. Immunohistochemistry of AT8 and Iba1 in the frontal cortex, dentate gyrus, and entorhinal cortex (E). Scale bar, 20 μm. Quantification of AT8 signal intensities (F) and the number of Iba1- positive cells (G). Two-way ANOVA with Tukey test, n = 7, 6, 3, 5. Data are represented as mean ± SEM. N.S., not significant.

Article Snippet: The primary antibodies used in the study: Tomaralimab (Amyloid Solution Inc., 1:250), anti-human TLR2 (Cell Signaling Technology #12276, 1:1000), anti-mouse TLR2 (Cell Signaling Technology #13744, 1:1000), anti-human tau HT7 (Invitrogen #MN1000, 1:1000), antiTau4R (Cell Signaling Technology #30328, 1:1000), anti-phospho-tau AT8 (Invitrogen #MN1020, 1:500), anti-Iba1 (GeneTex #GTX635400, 1:500), anti-Iba1 (Wako #019-19741, 1:1000), anti-MAP2 (Cell Signaling Technology #4542, 1:500), anti-NeuN (Millipore #MAB377, 1:1000), anti-GFAP (Cell Signaling Technology #80788, 1:1000), antiMyD88 (Cell Signaling Technology #50010, 1:1000), anti-NLRP3 (Cell Signaling Technology #15101, 1:1000), anti-β-actin (Santa Cruz Biotechnology #sc-47778, 1:1000), anti-GFP (Santa Cruz Biotechnology #sc-9996, 1:3000), anti-His-Tag (Cell Signaling Technology #2365, 1:1000).

Techniques: Knock-Out, RNA Expression, Activation Assay, Immunohistochemistry

Journal: Brain, behavior, and immunity

Article Title: TLR2 immunotherapy suppresses neuroinflammation, tau spread, and memory loss in rTg4510 mice.

doi: 10.1016/j.bbi.2024.08.002

Figure Lengend Snippet: Fig. 2. Microglial TLR2 binds to oligomeric tau. (A) Monomeric tau (mTau) and oligomeric tau (oTau) proteins prepared in vitro were subjected to native PAGE and stained with Coomassie Brilliant Blue or immunoblotted with anti-human tau (HT7) antibody. (B-C) TLR2 preferentially binds to oTau. Extracts of HEK293T cells transfected with TLR2-GFP were incubated without (None) or with either mTau or oTau (250 nM, 12 h) and subjected to immunoprecipitation (IP) (B). The signals of TLR2 on the blots were measured by ImageJ (C). One-way ANOVA with Holm-ˇSíd´ak test, n = 3. (D-E) Mapping of TLR2 domains involved in tau binding. A schematic diagram of TLR2 domains and mutants (ΔLBD, ΔDimer, D327W/F349L, P681H) (D). HEK293T cells were transfected with either WT or mutant TLR2-GFP. Cell lysates were incubated with 250 nM oTau and subjected to immunoprecipitation (IP) (E). (F-G) TLR2 activation increases binding of oligomeric tau. BV2 cells were left untreated (Nontreat) or incubated with Pam3CSK4 (10 μg/ml) overnight and then incubated with DyLight 488-labeled mTau or oTau (200 nM, 4 h). Cells were washed and immunostained with anti-TLR2 antibody (F). Scale bar, 10 μm. TLR2-bound DyLight 488 signals were measured by ImageJ (G). Two-way ANOVA with Tukey test, n = 3, 50 cells per group. Data are represented as mean ± SEM.

Article Snippet: The primary antibodies used in the study: Tomaralimab (Amyloid Solution Inc., 1:250), anti-human TLR2 (Cell Signaling Technology #12276, 1:1000), anti-mouse TLR2 (Cell Signaling Technology #13744, 1:1000), anti-human tau HT7 (Invitrogen #MN1000, 1:1000), antiTau4R (Cell Signaling Technology #30328, 1:1000), anti-phospho-tau AT8 (Invitrogen #MN1020, 1:500), anti-Iba1 (GeneTex #GTX635400, 1:500), anti-Iba1 (Wako #019-19741, 1:1000), anti-MAP2 (Cell Signaling Technology #4542, 1:500), anti-NeuN (Millipore #MAB377, 1:1000), anti-GFAP (Cell Signaling Technology #80788, 1:1000), antiMyD88 (Cell Signaling Technology #50010, 1:1000), anti-NLRP3 (Cell Signaling Technology #15101, 1:1000), anti-β-actin (Santa Cruz Biotechnology #sc-47778, 1:1000), anti-GFP (Santa Cruz Biotechnology #sc-9996, 1:3000), anti-His-Tag (Cell Signaling Technology #2365, 1:1000).

Techniques: In Vitro, Clear Native PAGE, Staining, Transfection, Incubation, Immunoprecipitation, Binding Assay, Mutagenesis, Activation Assay, Labeling

Journal: Brain, behavior, and immunity

Article Title: TLR2 immunotherapy suppresses neuroinflammation, tau spread, and memory loss in rTg4510 mice.

doi: 10.1016/j.bbi.2024.08.002

Figure Lengend Snippet: Fig. 4. Tau-induced TLR2 activation in microglia promotes neuronal tau uptake. (A-B) Microglial TLR2 increases neuronal tau uptake in neuron-microglia co- culture system. WT and Tlr2 KO mouse primary cortical neurons (DIV 7) and microglia (DIV 14) were co-cultured and treated with DyLight 488-oligomeric tau (200 nM, 24 h) (A). Scale bar, 10 μm. DyLight 488 intensities in MAP2-positive neurons (arrowheads) were measured (B). Two-way ANOVA with Tukey test, n = 3, 28–58 cells per group. (C-D) Tlr2 deficiency reduces tau-induced microglial activation in the mouse hippocampus. Tau oligomers (6 μg) were intracranially injected into the hippocampus of 4-month-old mice. After 7 days, brain sections were immunostained with anti-human tau (HT7) and anti-Iba1 antibodies (C). Scale bar, 50 μm. Manders’ colocalization coefficient (fraction of HT7 overlapping Iba1) was measured (D). Unpaired t-test, two-tailed, n = 3 per group. (E-F) Tlr2 deficiency reduces neuronal tau uptake in the mouse hippocampus. Tau oligomers (6 μg) were intracranially injected into the hippocampus of 3-month-old mice. After 48 h, brain sections were immunostained with anti-human tau (HT7) and anti-MAP2 antibodies (E). Arrowheads indicate internalized tau oligomers. Scale bar, 10 μm. Per- centages of HT7-positive neurons in the injection area were estimated (F). Unpaired t-test, two-tailed, n = 3 per group. Data are represented as mean ± SEM.

Article Snippet: The primary antibodies used in the study: Tomaralimab (Amyloid Solution Inc., 1:250), anti-human TLR2 (Cell Signaling Technology #12276, 1:1000), anti-mouse TLR2 (Cell Signaling Technology #13744, 1:1000), anti-human tau HT7 (Invitrogen #MN1000, 1:1000), antiTau4R (Cell Signaling Technology #30328, 1:1000), anti-phospho-tau AT8 (Invitrogen #MN1020, 1:500), anti-Iba1 (GeneTex #GTX635400, 1:500), anti-Iba1 (Wako #019-19741, 1:1000), anti-MAP2 (Cell Signaling Technology #4542, 1:500), anti-NeuN (Millipore #MAB377, 1:1000), anti-GFAP (Cell Signaling Technology #80788, 1:1000), antiMyD88 (Cell Signaling Technology #50010, 1:1000), anti-NLRP3 (Cell Signaling Technology #15101, 1:1000), anti-β-actin (Santa Cruz Biotechnology #sc-47778, 1:1000), anti-GFP (Santa Cruz Biotechnology #sc-9996, 1:3000), anti-His-Tag (Cell Signaling Technology #2365, 1:1000).

Techniques: Activation Assay, Co-Culture Assay, Cell Culture, Injection, Two Tailed Test

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: HRP2 increases pro-inflammatory and Toll-like receptor (TLR) genes in cortical organoids. HRP2-induced inflammation-related genes were profiled Fold changes greater than 1.5 and p values < 0.05 calculated using Student’s t -test was considered to be a significant dysregulation. (A) A heatmap was generated showing up-and down-regulated genes. The effects of HRP2 on cortical organoids at day 50 in culture are shown by site (B), including cerebral cortex and glia, as well as by pathway (C), including TLR signaling. Further analysis revealed the relationship between upregulated genes and activation of TLR2 pathway (D). Western blot densitometry of organoid lysates (data represented as means ± SEM) confirms that HRP2 significantly induces TLR2 and TLR1 expression, whereas NRG1 reduces these effects (E, F). a: p < 0.05 compared to basal conditions; b: p < 0.05 compared to HRP2 treatment. One-way ANOVA and t -test were used. Please also see Figure S4 .

Article Snippet: Rabbit monoclonal anti-human TLR2 (clone D769Z) , Cell Signaling , Cat #12276T RRID: AB_2797867.

Techniques: Generated, Activation Assay, Western Blot, Expressing

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: HRP2 uses TLR1 and TLR2 to increase CXCL10 expression and cell death in iPSC Cell proliferation, apoptosis, and expression of inflammatory markers were assessed in iPSC treated with HRP2 after TLR1 and TLR2 blockage. (A) Proliferation assay showed that IPSC growth was partially restored, whereas their apoptosis (B) and necrosis (C) were reduced to the levels of untreated controls. TLR1 and TLR2 blocking in HRP2-treated iPSC has an antiapoptotic and anti-inflammatory effect, as it is confirmed by IHC: the expression of cleaved caspase 3 (D, F) and CXCL10 (E, G) is reduced. Data represented as means ± SEM a: p < 0.05 compared to basal conditions; b: p < 0.05 compared to HRP2 treatment. Statistical significance between groups was determined using t -test and analysis of variance.

Article Snippet: Rabbit monoclonal anti-human TLR2 (clone D769Z) , Cell Signaling , Cat #12276T RRID: AB_2797867.

Techniques: Expressing, Proliferation Assay, Blocking Assay

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 TLR2 (clone D769Z) , Cell Signaling , Cat #12276T RRID: AB_2797867.

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

Journal: Cell Communication and Signaling : CCS

Article Title: Immune response in piglets orally immunized with recombinant Bacillus subtilis expressing the capsid protein of porcine circovirus type 2

doi: 10.1186/s12964-020-0514-4

Figure Lengend Snippet: The expression levels of Toll-like receptors (TLRs), IgA, and cytokines . Associated immune factors were measured in ileum homogenates prepared on day 39 of the experiment (at slaughter). a TLR2 protein expression, the ratios of TLR2 to GAPDH were normalized to the control. b TLR9 protein expression, the ratios of TLR9 to GAPDH were normalized to the control. c IgA expression. d-h Cytokine IL1β, IL-6, IL-10, TNF-α, and IFN-γ expression. i Porcine β-defensin 2 expression. The error bars represent standard deviations. * 0.01 < p < 0.05, ** p < 0.01 (compared to the Ctrl group)

Article Snippet: Rabbit anti-human TLR2 and TLR9 polyclonal antibodies were purchased from Thermo Scientific USA.

Techniques: Expressing