Journal: PLoS ONE

Article Title: Impact of cagPAI and T4SS on the Inflammatory Response of Human Neutrophils to Helicobacter pylori Infection

doi: 10.1371/journal.pone.0064623

Figure Lengend Snippet: The percentage of neutrophils expressing TLR9 3, 6, and 24 h after infection (A, B, and C, respectively) with cagPAI + strain 26695, T4SS mutants ( virB4 – and virD4 – ), and cagPAI – strain 8822. The data shown are from three independent experiments and line represent median. The results were obtained as medians ± intervals of percentiles and analyzed with the Kruskal–Wallis one-way test. Differences were considered statistically significant when *P<0.05. MC = mock control.

Article Snippet: Another set of neutrophils was incubated with PE-conjugated anti-human TLR2 and TLR4 (Santa Cruz Biotechnology, Santa Cruz, CA) and fluorescein-isothiocyanate-conjugated anti-human TLR5 (Santa Cruz Biotechnology) and anti-human TLR9 (Imgenex Corp., San Diego, CA).

Techniques: Expressing, Infection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Absent Toll-like receptor-9 expression predicts poor prognosis in renal cell carcinoma

doi: 10.1186/1756-9966-30-84

Figure Lengend Snippet: TLR9 immunostaining in RCC . Tumours with high cytoplasmic expression (A) and negative cytoplasmic expression (B) are shown. Magnification ×400, scale bar 50 μm.

Article Snippet: Expression of TLR9 was analyzed by using a mouse monoclonal anti-human TLR9/CD289 (Img-305A, clone 26C593.2, Imgenex, San Diego, California, USA, dilution 1:200) antibody, as previously shown by us [ , ].

Techniques: Immunostaining, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Absent Toll-like receptor-9 expression predicts poor prognosis in renal cell carcinoma

doi: 10.1186/1756-9966-30-84

Figure Lengend Snippet: Associations between cytoplasmic TLR9 expression and tumour pT-class, stage, grade and histological subtype

Article Snippet: Expression of TLR9 was analyzed by using a mouse monoclonal anti-human TLR9/CD289 (Img-305A, clone 26C593.2, Imgenex, San Diego, California, USA, dilution 1:200) antibody, as previously shown by us [ , ].

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Absent Toll-like receptor-9 expression predicts poor prognosis in renal cell carcinoma

doi: 10.1186/1756-9966-30-84

Figure Lengend Snippet: Associations between cytoplasmic TLR9 expression and RCC-specific survival . Patients with TLR9 negative tumours showed reduced survival when compared to patients with tumours positive for these proteins. p = 0.007

Article Snippet: Expression of TLR9 was analyzed by using a mouse monoclonal anti-human TLR9/CD289 (Img-305A, clone 26C593.2, Imgenex, San Diego, California, USA, dilution 1:200) antibody, as previously shown by us [ , ].

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Absent Toll-like receptor-9 expression predicts poor prognosis in renal cell carcinoma

doi: 10.1186/1756-9966-30-84

Figure Lengend Snippet: Cox multivariate survival analysis in 136 patients with RCC

Article Snippet: Expression of TLR9 was analyzed by using a mouse monoclonal anti-human TLR9/CD289 (Img-305A, clone 26C593.2, Imgenex, San Diego, California, USA, dilution 1:200) antibody, as previously shown by us [ , ].

Techniques: Expressing

Journal: PLoS ONE

Article Title: Spontaneous Production of Immunoglobulin M in Human Epithelial Cancer Cells

doi: 10.1371/journal.pone.0051423

Figure Lengend Snippet: A, TLR9 mRNA was detected in several cancer cell lines, HeLa MR, HeLa, HT-29, SW480, U-2 OS, 293 and 293T, by RT-PCR. Raji, as positive control; GAPDH, internal control. B, TLR9 expression was detected in HeLa MR cells by flow cytometry. Gray, isotype control IgG1; Black, anti-human TLR9. C, TLR9 expression was detected in HeLa MR cells by Western blot. Raji, as positive control; β-actin, internal control. D, expression of the TLR9-associated adapter MyD88 was detected in HeLa MR cells by RT-PCR. Raji, as positive control.

Article Snippet: The membranes were blocked with Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat milk or 5% bovine serum albumin (for detecting phosphoproteins) at room temperature for 2 hours, and were incubated with primary antibodies, such as mouse anti-human Ig µ mAb (Mabworks), goat anti-human Ig µ polyclonal antibody (Sigma, St. Louis, MO, USA), mouse anti-human CD79A and CD79B mAbs (Abcam, Cambridge, MA, USA), mouse anti-human TLR9 mAb (Imgenex), anti-phospho-PKC mAb, anti-phospho-Akt mAb, anti-phospho-ERK mAb, anti-phospho-IκB mAb, and anti-IκB mAb (all from Cell Signaling, Danvers, MA, USA), rabbit anti-ERK polyclonal antibody (KangCheng Biotech Co., Shanghai, China), and anti-actin mAb (Sigma), at 4°C overnight.

Techniques: Reverse Transcription Polymerase Chain Reaction, Positive Control, Expressing, Flow Cytometry, Western Blot

Journal: PLoS ONE

Article Title: Spontaneous Production of Immunoglobulin M in Human Epithelial Cancer Cells

doi: 10.1371/journal.pone.0051423

Figure Lengend Snippet: A, Ig µ mRNA level was analyzed by semiquantitative RT-PCR after stimulation by CpG 2006, and the two non-CpG ODN controls, CpG 2078 and GpC. CpG-N ODN208, as negative control; GAPDH, internal control. B, flow cytometry analysis showed that cytoplasmic IgM was decreased after stimulation with CpG 2006, CpG 2078, and GpC. CpG-N ODN208, as negative control. ** P <0.01 vs. vehicle control. C, Western blot analysis showed that cytoplasmic IgM was decreased after stimulation with CpG 2006, CpG 2078, and GpC. CpG-N ODN208, as negative control; β-actin, internal control. D, Western blot analysis showed increased level of secreted IgM after stimulation with CpG 2006, CpG 2078, and GpC. CpG-N ODN208, as negative control. E, phosphorylation of ERK, a molecule downstream of TLR9 signaling cascade, was detected by Western blot after stimulation with CpG 2006, CpG 2078, and GpC. EGF, epidermal growth factor was used as a positive control for ERK phosphorylation. F, Western blot analysis showed that IκB, a molecule in NF-κB pathway, also was phosphorylated after stimulation with CpG 2006, CpG, 2078 and GpC.

Article Snippet: The membranes were blocked with Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat milk or 5% bovine serum albumin (for detecting phosphoproteins) at room temperature for 2 hours, and were incubated with primary antibodies, such as mouse anti-human Ig µ mAb (Mabworks), goat anti-human Ig µ polyclonal antibody (Sigma, St. Louis, MO, USA), mouse anti-human CD79A and CD79B mAbs (Abcam, Cambridge, MA, USA), mouse anti-human TLR9 mAb (Imgenex), anti-phospho-PKC mAb, anti-phospho-Akt mAb, anti-phospho-ERK mAb, anti-phospho-IκB mAb, and anti-IκB mAb (all from Cell Signaling, Danvers, MA, USA), rabbit anti-ERK polyclonal antibody (KangCheng Biotech Co., Shanghai, China), and anti-actin mAb (Sigma), at 4°C overnight.

Techniques: Reverse Transcription Polymerase Chain Reaction, Negative Control, Flow Cytometry, Western Blot, Positive Control

Journal: PLoS ONE

Article Title: Spontaneous Production of Immunoglobulin M in Human Epithelial Cancer Cells

doi: 10.1371/journal.pone.0051423

Figure Lengend Snippet: A, semi-quantative RT-PCR showed increased Ig µ expression after stimulation by CpG 2006, CpG 2078 and GpC (upper panel). The effects of TLR9 agonists were abolished by chloroquine (lower panel). GAPDH, internal control. B, flow cytometry analysis showed that the cytoplasmic IgM level was decreased after stimulation with CpG 2006, CpG 2078 and GpC, and that this was abolished by chloroquine. * P <0.05 vs. vehicle control; ** P <0.01 vs. vehicle control. C, Western blot analysis showed that the cytoplasmic IgM level was decreased after stimulation with CpG 2006, CpG 2078 and GpC, and that this was abolished by chloroquine. D, Western blot analysis showed that IκB was phosphorylated after stimulation with CpG 2006, CpG 2078 and GpC, and that this was abolished by chloroquine.

Article Snippet: The membranes were blocked with Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat milk or 5% bovine serum albumin (for detecting phosphoproteins) at room temperature for 2 hours, and were incubated with primary antibodies, such as mouse anti-human Ig µ mAb (Mabworks), goat anti-human Ig µ polyclonal antibody (Sigma, St. Louis, MO, USA), mouse anti-human CD79A and CD79B mAbs (Abcam, Cambridge, MA, USA), mouse anti-human TLR9 mAb (Imgenex), anti-phospho-PKC mAb, anti-phospho-Akt mAb, anti-phospho-ERK mAb, anti-phospho-IκB mAb, and anti-IκB mAb (all from Cell Signaling, Danvers, MA, USA), rabbit anti-ERK polyclonal antibody (KangCheng Biotech Co., Shanghai, China), and anti-actin mAb (Sigma), at 4°C overnight.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Flow Cytometry, Western Blot

Journal: Mediators of Inflammation

Article Title: Impaired Surface Expression of HLA-DR, TLR2, TLR4, and TLR9 in Ex Vivo-In Vitro Stimulated Monocytes from Severely Injured Trauma Patients

doi: 10.1155/2017/2608349

Figure Lengend Snippet: Whole blood from healthy volunteers (HV, n = 14) or major trauma patients (TP, n = 29) was analyzed over a 10-day time course after admission (emergency department, ED–10) by flow cytometry. Monocytes were detected using anti-human CD14 in the corresponding sideward and forward scatter. Unstimulated (black symbols) and stimulated (clear symbols) measurements were made. For stimulation (stim), whole blood was incubated with leukocyte activation cocktail for 5 h with subsequent analyzing procedure as in unstimulated samples. Data are shown as mean ± SEM. (a) TLR2 expression in CD14 + monocytes, (b) TLR4 expression in CD14 + monocytes, (c) TLR9 expression in CD14 + monocytes, (d) TLR2 expression in CD14 + HLA-DR − monocytes, (e) TLR4 expression in CD14 + HLA-DR − monocytes, and (f) TLR9 expression in CD14 + HLA-DR − monocytes. ∗ p < 0.05 versus unstimulated HV; ⌀ p < 0.05 versus stimulated HV; æ p < 0.05 TP versus corresponding TP stim; # p < 0.05 HV ctrl versus all; & p < 0.05 versus all; ∞ p < 0.05 TP stim versus corresponding unstimulated TP and HV stim.

Article Snippet: Blood samples (100 μ l) were transferred into polystyrene FACS tubes (BD Pharmingen) and incubated with mouse anti-human CD14 V500 (Clone M5E2, BD Bioscience, San Jose, CA), mouse anti-human TLR2 PE-Cy7 (Clone T2.5, eBioscience, San Diego, CA), mouse anti-human TLR4 FITC (Clone HTA125, IMGENEX, San Diego, CA), mouse anti-human TLR9 Alexa Flour® 647 (Clone 26C593.2, IMGENEX, San Diego, CA), and mouse anti-human HLA-DR PerCP-Cy5.5 conjugated (Clone L243, BD Bioscience, San Jose, CA) antibodies.

Techniques: Flow Cytometry, Incubation, Activation Assay, Expressing

Journal: Mediators of Inflammation

Article Title: Impaired Surface Expression of HLA-DR, TLR2, TLR4, and TLR9 in Ex Vivo-In Vitro Stimulated Monocytes from Severely Injured Trauma Patients

doi: 10.1155/2017/2608349

Figure Lengend Snippet: Whole blood from healthy volunteers (HV, n = 14) or major trauma patients (TP, n = 29) was analyzed by flow cytometry over a 10-day time course after admission (emergency department, ED–10). Monocytes were detected using anti-human CD14 in the corresponding sideward and forward scatter. Unstimulated (black symbols) and stimulated (clear symbols) measurements were made. For stimulation (stim), whole blood was incubated with leukocyte activation cocktail for 5 h with subsequent analyzing procedure as in unstimulated samples. Data are shown as mean ± SEM. (a) TLR2/HLA-DR coexpression on CD14 + monocytes, (b) TLR4/HLA-DR coexpression on CD14 + monocytes, and (c) TLR9/HLA-DR coexpression on CD14 + monocytes. ∗ p < 0.05 versus unstimulated HV; æ p < 0.05 TP versus corresponding TP stim; & p < 0.05 versus all; ∞ p < 0.05 TP stim versus corresponding unstimulated TP and HV stim.

Article Snippet: Blood samples (100 μ l) were transferred into polystyrene FACS tubes (BD Pharmingen) and incubated with mouse anti-human CD14 V500 (Clone M5E2, BD Bioscience, San Jose, CA), mouse anti-human TLR2 PE-Cy7 (Clone T2.5, eBioscience, San Diego, CA), mouse anti-human TLR4 FITC (Clone HTA125, IMGENEX, San Diego, CA), mouse anti-human TLR9 Alexa Flour® 647 (Clone 26C593.2, IMGENEX, San Diego, CA), and mouse anti-human HLA-DR PerCP-Cy5.5 conjugated (Clone L243, BD Bioscience, San Jose, CA) antibodies.

Techniques: Flow Cytometry, Incubation, Activation Assay

Journal: European Journal of Histochemistry : EJH

Article Title: Evaluation of expression of Toll-Like Receptors 7 and 9, proliferation, and cytoskeletal biomarkers in plaque and guttate psoriasis: A pilot morphological study

doi: 10.4081/ejh.2021.3218

Figure Lengend Snippet: TLR7 and TLR9 immunostaining in paraffin skin sections. Representative TLR7 (a-d) and TLR9 (e-h) immunostainings in non lesional and lesional skin of PV and PG. a,e) Non lesional PV; b,f ) lesional PV; c,g) non lesional PG; d,h) lesional PG. TLR7, Tolllike Receptor 7; TLR9, Toll-like Receptor 9; PV, psoriasis vulgaris; PG, guttate psoriasis. White dotted line indicates the basal membrane. Scale bars: a-d) 50 μm; e-h) 25 μm.

Article Snippet: After unspecific binding site saturation with 10% normal goat serum (Vector Laboratories) in 0.1 M PBS (pH 7.4; 30 min at RT), the following primary antibodies were used: rabbit anti-human TLR7 (Novus Biologicals, Centennial, Co, USA; dilution 1:300 in 0.1 M PBS/BSA 2%; overnight at 4°C), mouse anti-human TLR9 (Novus Biologicals; dilution 1:10 in 0.1 M PBS; overnight at 4°C), rabbit anti-human K17 (Abcam, Cambridge, UK; dilution 1:200 in 0.1 M PBS/BSA 1%; overnight at 4°C ), mouse anti-human K10 (Santa Cruz Biotechnology; dilution 1:50 in 0.1 M PBS/BSA 1%; overnight at 4°C), and rabbit anti-human K16 (Bio SB, Santa Barbara, CA, USA; dilution 1:100 in 0.1 M PBS/BSA 1%; 1 h at 37°C).

Techniques: Immunostaining