14-9922-80

antibody from Invitrogen Antibodies

Targeting: TLR2 CD282, TIL4

Provider product page for 14-9922-80

Western blot

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [62]
Comments [0]
Validations
Flow cytometry [1]
Other assay [9]

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Product number: 14-9922-80 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD282 (TLR2) Monoclonal Antibody (TL2.1), eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The TL2.1 monoclonal antibody reacts with human Toll-like receptor 2 (TLR2). To date, at least ten members of the Toll family have been identified in human. This family of type I transmembrane proteins is characterized by an extracellular domain with leucine-rich repeats and a cytoplasmic domain with homology to the type I IL-1 receptor. Two of these receptors, TLR2 and TLR4, are pattern recognition receptors and signaling molecules in response to bacterial lipoproteins and have been implicated in innate immunity and inflammation. TLR2 is expressed by peripheral blood monocytes and is responsible for distinguishing different pathogens. TL2.1, a blocking antibody, has been used to study the role of TLR-2 as a pattern recognition receptor in microbial lipoprotein/lipopeptide induced cytokine production from human peripheral blood mononuclear cells. TL2.1 has been reported to immunoprecipitate human TLR2 (~90 kDa) from PBMC and HMEC. Applications Reported: The TL2.1 antibody has been reported for use in flow cytometric analysis, immunoprecipitation, immunoblotting (WB), and immunohistochemical staining of frozen as well as paraffin embedded tissue sections. It has also been reported in blocking of TLR2-mediated cytokine production. The reported applicability of TL2.1 for immunoblotting and histochemistry may require high expression levels of TLR2 and has not been confirmed in our tests. (Please use Functional Grade purified TL2.1, Product # 16-9922, in functional assays). Applications Tested: The TL2.1 antibody has been tested by flow cytometric analysis of normal human peripheral blood cells and can be used at less than or equal to 2 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Purity: Greater than 90%, as determined by SDS-PAGE. Aggregation: Less than 10%, as determined by HPLC. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: TL2.1
Vial size: 25 µg
Concentration: 0.5 mg/mL
Storage: 4° C

TLR2 protein structure - 14-9922-80 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 9 Interleukin-1beta plays a key role in the downregulation of RCT mediators by hepatocytes in response to products secreted by activated hepatic macrophages. (A,B) Human HepG2 hepatocytes increased serum amyloid A (SAA)-1, and reduced ApoAI, mRNA expression significantly in response to IL-1beta, but not stimulants of NOD1 (iEDAP), TLR2 (Pam 3 CSK 4 ), or TLR4 ( E. coli LPS). (C) Conditioned medium of HepG2 cells significantly increased the efflux of 3 H-radiolabelled cholesterol from J774 macrophages. (D) The capacity of conditioned media of HepG2 hepatocytes to accept 3 H-radiolabelled cholesterol effluxed from J774 macrophages was reduced significantly by IL-1beta, but not by IL-6, TNF-alpha, or stimulants of NOD1, TLR2, or TLR4. (E) Flow cytometry showed abundant surface TLR2 and TLR4 on human primary monocytes, but no TLR2 and little TLR4 on HepG2 hepatocytes. Murine primary hepatocytes isolated by hepatic perfusion also showed no expression of TLR2 or TLR4, in low-fat diet (LFD) or high-fat diet (HFD)-primed mice (representative of 4 experiments). (F) Kinetics of ApoAI protein expression in HepG2 cells treated with IL-1beta determined by Western blot. (G) HepG2 ApoAI mRNA response to conditioned medium of LPS-treated human monocytes with isotype-control or IL-1beta neutralizing antibody ( n = 5 exps). (H,I) ApoAI and SAA1 mRNA responses of primary hepatocytes (isolated from n = 6 LFD-fed mice) to inflammatory cytokines (20 ng/ml). (J) ApoAI mRNA responses of prima

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Characterization of TLR2 binding proteins from Leptospira . ( A ) Immuno-precipitant of purified TLR2 protein and Loa22 protein. Anti-TLR2, anti-LipL32 and anti-Loa22 antibodies were used to detect the presence of these proteins. Lane 1: anti-V5 antibody activated Protein A beads incubated with HEK293-TLR2 cell lysate; lane 2: anti-V5 antibody activated Protein A beads incubated with LOMP; lane 3: LOMP; lane 4: Co-IP of TLR2 and LOMP fractions. ( B ) The expression of Loa22 protein in different species and fractions of Leptospira . Recombinant Loa22 (rLoa22) was used as positive control (lane 1). Peptidoglycan from L. santarosai (LPGN) was also used to detect the presence of Loa22 protein (lane 2). LOMPs from different species of Leptospires were used to detect the presence or absence of Loa22 protein including pathogenic L. interrogans (lane 3), non-pathogenic L. biflexa (lane 4), and L. santarosai (lane 5). Rabbit polyclone anti-Loa22 antibody was use to recognize the protein. ( C ) Purification of the Loa22 and mutation variants. Wild type and mutation variants were analyzed by SDS-PAGE (lower panel) and Western blot (upper panel; recognized by anti 6XHis tag antibody) Lane 1: WT Loa222; lane 2; Loa22D122A; lane 3: Loa22R143A. ( D ) Size exclusion chromatography of purified Loa22 protein. The single peak of purified Loa22 protein with molecular mass about 22 kDa indicated that the protein was of uniform conformation. The standard markers were ferritin (440 kDa), b

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Loa22 co-localized with TLR2 on HEK293-TLR2 cells. HEK293-TLR2 cell were cultured to 70% confluence and serum free for 16 h before adding the stimulation agents (0.1 mug/ml). The cells were incubated with the stimulation agents for 4 h and then fixed to against with the anti-V5 antibody (1:5000), anti-Loa22 antibody (1:10,000). The relative Alex488 and Alex594 conjugated secondary antibodies were used to stained TLR2 and Loa22 proteins, respectively. ( A ) rLoa22WT was incubated with HEK293 cell. ( B ) rLoa22WT-LPGN was incubated with HEK293-TLR2 cell. ( C ) rLoa22D122A-LPGN was incubated with HEK293-TLR2 cell. ( D ) rLoa22R143A-LPGN was incubated with HEK293-TLR2 cell. ( E ) Pam 3 CSK 4 Rhodamine was incubated with HEK293-TLR2 cell. The nucleus was stained by DAPI (blue) and TLR2 was stained by Alexa488 (green). Loa22 was stained by Alexa594 (red). The yellow color indicated that the two proteins were co-localized in HEK293-TLR2 cell.

Supportive validation

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Experimental details: 10.1371/journal.pone.0208940.g004 Fig 4 HSP90B1 knock down does not alter TLR2 surface expression or Mtb replication. Using CRISPR/Cas9, HSP90B1 was targeted for gene deletion in U937 macrophage-like human cells. A: Confirmation of knockout. Restriction fragment length polymorphism (RFLP) verified the loss of the ApaI restriction endonuclease site in the targeted lines (KO) but not the wild type (WT) nor cell line exposed to the CRISPR apparatus with a nonspecific guide sequence (EV). Western blot revealed reduction of the expected band in the KO line (b-actin as loading control). B: Wildtype or HSP90B1 knock down cell lines were stained with anti-TLR2 antibody or isotype control (iso) and evaluated for surface expression using flow cytometry. C: Knock down or control cell lines were infected with Erdman strain Mtb constitutively expressing luciferase at a multiplicity of infection of 5. Relative light units (RLUs) act as a proxy for colony count in this assay. Daily RLUs were measured from day 3 to day 7. Included is a background control where the Mtb was added to wells in the absence of human cells (mLux). Each exposure was repeated in sextuplet and the mean and standard deviation are provided for each cell line and time point.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 1 Sustained high expression of TLR2 and increased frequency of monocytes correlates with DENV disease severity. a - e PBMCs were isolated from 15 age-matched healthy donors (HD) and 54 patients undergoing acute DENV infection (DENV+) who developed relatively mild (DF, n = 32) or severe (DHF/DSS, n = 22) disease. a Monocyte subsets distribution in healthy and DENV+ patients (two-tailed Mann-Whitney test, **** P < 0.0001). b Percentages of cells expressing TLR2 were determined for each monocyte subset (two-tailed Mann-Whitney test, **** P < 0.0001) and c stratified by disease severity (two-tailed Mann-Whitney test, * P < 0.05; ** P < 0.01). d Percentages of NS3+ infected cells in DENV-positive patients ( n = 15) stained intracellularly for DENV NS3 (two-tailed Mann-Whitney test, *** P < 0.001; **** P < 0.0001). e Monocyte subsets distribution in patients, stratified by disease severity (two-tailed Mann-Whitney test, * P < 0.05). CM classical monocytes, IM intermediate monocytes, NM non-classical monocytes. Bars represent median with interquartile range (IQR). Source data are provided as a Source data file.

Supportive validation

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Experimental details: Figure 1. TLR2 expression in tumor and the peritumoral tissues from HCC patients. (A) Quantitative PCR, (B) flow cytometry and (C) western blot analysis of TLR2 expression. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. (A) TLR2 expression in the B76/Huh7 cell line at 48 h following culture with the serum of patients with HCC (Ca), the serum of healthy individuals (normal) or FBS by flow cytometry. (B) IL-6, IL-8, interferon (INF)-beta and tumor necrosis factor (TNF)-alpha in B76/Huh7 cell line 48 h after culture with the serum of patients with hepatocellular carcinoma, with the serum of healthy individuals or FBS. Reverse transcription-quantitative PCR was performed with the fold change of IL-6 in the cells supplemented with FBS as the positive internal control. The data for the samples are expressed relative to the average value of the control samples +- standard deviations of the means. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 Characterization of EP2-positive immune cells. a - d Peripheral whole blood (at least 5 subjects per group) was stained with anti-CD14, anti-CD16, anti-EP2, anti-TLR2, anti-HLA-DR, anti-CD11b or anti-TLR4. The percentages of different phenotypes of EP2 + or EP2 - monocytes and neutrophils were determined by flow cytometry. e PBMCs isolated from at least 3 subjects per group were stimulated with LPS (100 ng/ml) for 72 h. Cytokine production was measured by flow cytometry. f Peripheral whole blood from 7 subjects per group was incubated with E. coli for 30 min. ROS production in neutrophils and monocytes was assessed by flow cytometry. The data are expressed as the mean +- SEM of individual subjects or plots of individual data. The error bar represented SEM and the horizontal line represented the median. Statistical analyses were analyzed using the Wilcoxon signed-rank test. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not statistically significant