mouse anti human tlr5  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: Stenotrophomonas maltophilia provokes NEU1-mediated release of a flagellin-binding decoy receptor that protects against lethal infection

doi: 10.1016/j.isci.2024.110866

Figure Lengend Snippet: Sm flagellin binds to the MUC1-ED (A) HEK293T cells were transfected with the pcDNA3.1 empty vector, or plasmids encoding for TLR5 (pTLR5) or MUC1 (pMUC1), the cells were lysed and the lysates processed for TLR5 (lanes 1, 2) or MUC1 (lanes 3, 4) immunoblotting. To control for protein loading and transfer, the blots were stripped and reprobed (IB∗) for β-tubulin. (B) HEK293T cells overexpressing TLR5 or MUC1, or empty vector-transfected cells, were incubated with Sm and adherent bacteria quantified. Vertical bars represent mean ± SEM CFUs/well. (C) A549 cells were transfected with pcDNA3.1 or pMUC1 and binding of increasing concentrations of the Alexa Fluor 594-labeled Sm FliC1,2,3 flagellin mixture determined. Data points represent mean ± SEM bound flagellin in molecules/EC. (D) Scatchard analysis of binding data in (C). The linear regression equation and R 2 value are indicated adjacent to each line. (E and F) HEK293T cells were infected with Ad-GFP or Ad-MUC1, and A549 cells were transfected with MUC1-targeting or control siRNAs, and both cell types were cultured for 48 h. (E) Ad-GFP- and Ad-MUC1-infected cells (lanes 1, 2) and MUC1 siRNA- and control siRNA-transfected cells (lanes 3, 4) were lysed and the lysates processed for MUC1 immunoblotting. (F) Infected or transfected cells were incubated with Sm and cell-bound bacteria quantified. Vertical bars represent mean ± SEM Sm adhesion in CFUs/well. (G) A549 cells were transfected with MUC1-targeting or control siRNAs, cultured for 48 h, and binding of increasing concentrations of the Alexa Fluor 595-labeled Sm FliC1,2,3 flagellin mixture determined. Data points represents mean ± SEM bound flagellin in molecules/EC. (H) Scatchard analysis of binding data in (G). (I–K) A549 cells and A549 cells transfected with MUC1-targeting or control siRNAs were incubated for 48 h and then stimulated for 1 h with 10 ng of the Sm FliC1,2,3 flagellin mixture or PBS. (I) Non-transfected, PBS-treated (lane 1), control siRNA-transfected, flagellin-stimulated (lane 2), and MUC1 siRNA-transfected, flagellin-stimulated (lane 3) cells were lysed, and the lysates processed for pERK1/2 immunoblotting. To control for protein loading and transfer, the blots were stripped and reprobed for total ERK2. The ∼44.0 kDa pERK1 (upper) and ∼42.0 kDa pERK2 (lower) bands, as well as the total ERK2 band, are indicated by arrowheads on the right. (J) Densitometric analysis of pERK1/2 normalized to total ERK2 displayed in (I). Vertical bars represent mean ± SEM normalized pERK1/2 signal. (K) Supernatants from the cells in (I) were processed for IL-8 levels and normalized to total EC protein. Vertical bars represent mean ± SEM normalized IL-8 concentrations in pg/mg EC protein. ∗, increased Sm adhesion (B, F), flagellin binding (C), ERK activation (J), or normalized IL-8 release (K) compared to pcDNA3.1, pTLR5, Ad-GFP, control siRNA, or PBS controls at p < 0.05. ∗∗, decreased Sm adhesion (F), flagellin binding (G), ERK activation (J), or normalized IL-8 release (K) compared to the siRNA controls at p < 0.05. n.s., not significant. Statistical comparisons were made using the Student’s t test. The results are representative of 3 or 6 independent experiments.

Article Snippet: Mouse anti-human TLR5 , Santa Cruz Biotechnology , Cat# sc-52963; RRID: AB_793184.

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Incubation, Bacteria, Binding Assay, Labeling, Infection, Cell Culture, Activation Assay

Journal: iScience

Article Title: Stenotrophomonas maltophilia provokes NEU1-mediated release of a flagellin-binding decoy receptor that protects against lethal infection

doi: 10.1016/j.isci.2024.110866

Figure Lengend Snippet:

Article Snippet: Mouse anti-human TLR5 , Santa Cruz Biotechnology , Cat# sc-52963; RRID: AB_793184.

Techniques: Virus, Recombinant, Control, Software, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Effects of Supplemental Calcium and Vitamin D on Expression of Toll-Like Receptors and Phospho-IKKα/β in the Normal Rectal Mucosa of Colorectal Adenoma Patients

doi: 10.1158/1940-6207.CAPR-18-0123

Figure Lengend Snippet: pIKKα/β, TLR4, and TLR5 Expression in the Full Length of Crypts and Stroma in the Normal-Appearing Rectal Mucosa of the Adjunct Biomarker Study Participants (n = 105) During the Trial a .

Article Snippet: Next, slides were immunohistochemically processed in a DakoCytomation Autostainer Plus System (Agilent Dako, Santa Clara, CA) automated immunostainer, using a labeled streptavidin-biotin kit (ThermoScientific UltraVisionKit, TP-125-HL, Fremont, CA), by applying a rabbit monoclonal antibody against pIKKα/β (catalog no.: 2697P, dilution, 1:150; Cell Signaling, Danvers, MA), a mouse monoclonal antibody against TLR4 (catalog no.: ab47093, dilution 1:325; Abcam, Cambridge, UK), and a mouse monoclonal antibody against TLR5 (catalog no.: MAB6704, dilution 1:200; R&D Systems, Minneapolis, MN).

Techniques: Expressing, Biomarker Discovery

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Effects of Supplemental Calcium and Vitamin D on Expression of Toll-Like Receptors and Phospho-IKKα/β in the Normal Rectal Mucosa of Colorectal Adenoma Patients

doi: 10.1158/1940-6207.CAPR-18-0123

Figure Lengend Snippet: Proportional Differences in pIKKα/β, TLR4, and TLR5 Expression in the Full Length of Crypts and Stroma, by Categories of Baseline Participant Characteristics (n = 105) a .

Article Snippet: Next, slides were immunohistochemically processed in a DakoCytomation Autostainer Plus System (Agilent Dako, Santa Clara, CA) automated immunostainer, using a labeled streptavidin-biotin kit (ThermoScientific UltraVisionKit, TP-125-HL, Fremont, CA), by applying a rabbit monoclonal antibody against pIKKα/β (catalog no.: 2697P, dilution, 1:150; Cell Signaling, Danvers, MA), a mouse monoclonal antibody against TLR4 (catalog no.: ab47093, dilution 1:325; Abcam, Cambridge, UK), and a mouse monoclonal antibody against TLR5 (catalog no.: MAB6704, dilution 1:200; R&D Systems, Minneapolis, MN).

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR stimulation of prostate tumor cells induces chemokine-mediated recruitment of specific immune cell types.

doi: 10.4049/jimmunol.0902401

Figure Lengend Snippet: FIGURE 1. TLR mRNA and protein expression in human PCa cell lines LNCaP, PC3, and DU-145. A, RT-PCR showing basal expression of the in- dicated Tlr genes. RT-PCR was performed using specific primers as de- scribed in Materials and Methods. B, Western blot analysis of TLR3 expression. Similar results were observed in three independent experiments (A, B). C, Flow cytometric analysis of TLR5 expression was performed using mouse anti-TLR5 mAb (white areas) or isotypic mouse IgG (gray areas). The diagrams are representative of at least three independent experiments.

Article Snippet: Mouse IgG2a anti-human TLR5 (Imgenex, San Diego, CA) mAb or the appropriate isotypic control mAb was used at 0.5 mg/106 cells for 30 min on ice.

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

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR stimulation of prostate tumor cells induces chemokine-mediated recruitment of specific immune cell types.

doi: 10.4049/jimmunol.0902401

Figure Lengend Snippet: FIGURE 4. Stimulation with agonists of TLR3 and TLR5 modulate chemokine production. LNCaP or DU-145 cells were treated for 24 h with poly(I:C) or flagellin, CM were collected, and ELISA assay was performed as described in Materials and Methods. Data represent the mean 6 SEM of quadruplicate samples from two independent experiments. Stat- istical analysis was performed by comparing che- mokine levels in TLR-stimulated CM versus control CM, when detectable. pp # 0.05; ppp # 0.01, Stu- dent t test. ND, not detectable.

Article Snippet: Mouse IgG2a anti-human TLR5 (Imgenex, San Diego, CA) mAb or the appropriate isotypic control mAb was used at 0.5 mg/106 cells for 30 min on ice.

Techniques: Enzyme-linked Immunosorbent Assay, Control