TLR4 Antibody - #AF7017

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Product: TLR4 Antibody
Catalog: AF7017
Description: Rabbit polyclonal antibody to TLR4
Application: WB IHC IF/ICC
Reactivity: Human, Mouse, Rat
Mol.Wt.: 100~130kDa; 96kD(Calculated).
Uniprot: O00206
RRID: AB_2835322

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MSDS
Data Sheet
COA
Protocols
WB Handbook
IHC Protocol
IF/ICC Protocol

Product Info

Source:
Rabbit
Application:
WB 1:500-1:2000, IHC 1:50-1:200, IF/ICC 1:100-1:500
*The optimal dilutions should be determined by the end user.
*Tips:

WB: For western blot detection of denatured protein samples. IHC: For immunohistochemical detection of paraffin sections (IHC-p) or frozen sections (IHC-f) of tissue samples. IF/ICC: For immunofluorescence detection of cell samples. ELISA(peptide): For ELISA detection of antigenic peptide.

Reactivity:
Human,Mouse,Rat
Clonality:
Polyclonal
Specificity:
TLR4 Antibody detects endogenous levels of total TLR4.
RRID:
AB_2835322
Cite Format: Affinity Biosciences Cat# AF7017, RRID:AB_2835322.
Conjugate:
Unconjugated.
Purification:
The antiserum was purified by peptide affinity chromatography using SulfoLink™ Coupling Resin (Thermo Fisher Scientific).
Storage:
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol. Store at -20 °C. Stable for 12 months from date of receipt.
Alias:

Fold/Unfold

ARMD10; CD284; CD284 antigen; Homolog of Drosophila toll; hToll; TLR 4; TLR4; TLR4_HUMAN; TOLL; Toll like receptor 4; Toll-like receptor 4;

Immunogens

Immunogen:
Uniprot:

O00206(TLR4_HUMAN) >>Visit HPA database.

Gene(ID):
TLR4(7099)
Expression:
O00206 TLR4_HUMAN:

Highly expressed in placenta, spleen and peripheral blood leukocytes (PubMed:9435236, PubMed:9237759). Detected in monocytes, macrophages, dendritic cells and several types of T-cells (PubMed:9237759, PubMed:27022195).

Description:
Toll like receptors (TLR) are highly conserved throughout evolution and have been implicated in the innate defense to many pathogens. In Drosophila toll is required for the anti fungal response, while the related 18 wheeler is involved in antibacterial defenses. In mammals, TLR identified as type I transmembrane signaling receptors with pattern recognition capabilities have been implicated in the innate host defense to pathogens. TLR4 has been identified next to MD2 and CD14 as a receptor that is central to the innate immune response to lipopolysaccharides (LPS) of Gram negative bacteria.
Sequence:
MMSASRLAGTLIPAMAFLSCVRPESWEPCVEVVPNITYQCMELNFYKIPDNLPFSTKNLDLSFNPLRHLGSYSFFSFPELQVLDLSRCEIQTIEDGAYQSLSHLSTLILTGNPIQSLALGAFSGLSSLQKLVAVETNLASLENFPIGHLKTLKELNVAHNLIQSFKLPEYFSNLTNLEHLDLSSNKIQSIYCTDLRVLHQMPLLNLSLDLSLNPMNFIQPGAFKEIRLHKLTLRNNFDSLNVMKTCIQGLAGLEVHRLVLGEFRNEGNLEKFDKSALEGLCNLTIEEFRLAYLDYYLDDIIDLFNCLTNVSSFSLVSVTIERVKDFSYNFGWQHLELVNCKFGQFPTLKLKSLKRLTFTSNKGGNAFSEVDLPSLEFLDLSRNGLSFKGCCSQSDFGTTSLKYLDLSFNGVITMSSNFLGLEQLEHLDFQHSNLKQMSEFSVFLSLRNLIYLDISHTHTRVAFNGIFNGLSSLEVLKMAGNSFQENFLPDIFTELRNLTFLDLSQCQLEQLSPTAFNSLSSLQVLNMSHNNFFSLDTFPYKCLNSLQVLDYSLNHIMTSKKQELQHFPSSLAFLNLTQNDFACTCEHQSFLQWIKDQRQLLVEVERMECATPSDKQGMPVLSLNITCQMNKTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI

PTMs - O00206 As Substrate

Site PTM Type Enzyme
N35 N-Glycosylation
N173 N-Glycosylation
T175 Phosphorylation
N205 N-Glycosylation
N282 N-Glycosylation
N309 N-Glycosylation
N497 N-Glycosylation
N526 N-Glycosylation
Y551 Phosphorylation
N575 N-Glycosylation
N624 N-Glycosylation
Y674 Phosphorylation
Y680 Phosphorylation
S730 Phosphorylation
S738 Phosphorylation
S790 Phosphorylation Q15139 (PRKD1)
S800 Phosphorylation

Research Backgrounds

Function:

Cooperates with LY96 and CD14 to mediate the innate immune response to bacterial lipopolysaccharide (LPS). Acts via MYD88, TIRAP and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response. Also involved in LPS-independent inflammatory responses triggered by free fatty acids, such as palmitate, and Ni(2+). Responses triggered by Ni(2+) require non-conserved histidines and are, therefore, species-specific. Both M.tuberculosis HSP70 (dnaK) and HSP65 (groEL-2) act via this protein to stimulate NF-kappa-B expression. In complex with TLR6, promotes sterile inflammation in monocytes/macrophages in response to oxidized low-density lipoprotein (oxLDL) or amyloid-beta 42. In this context, the initial signal is provided by oxLDL- or amyloid-beta 42-binding to CD36. This event induces the formation of a heterodimer of TLR4 and TLR6, which is rapidly internalized and triggers inflammatory response, leading to the NF-kappa-B-dependent production of CXCL1, CXCL2 and CCL9 cytokines, via MYD88 signaling pathway, and CCL5 cytokine, via TICAM1 signaling pathway, as well as IL1B secretion. Binds electronegative LDL (LDL(-)) and mediates the cytokine release induced by LDL(-). Stimulation of monocytes in vitro with M.tuberculosis PstS1 induces p38 MAPK and ERK1/2 activation primarily via TLR2, but also partially via this receptor.

PTMs:

N-glycosylated. Glycosylation of Asn-526 and Asn-575 seems to be necessary for the expression of TLR4 on the cell surface and the LPS-response. Likewise, mutants lacking two or more of the other N-glycosylation sites were deficient in interaction with LPS.

Phosphorylated on tyrosine residues by LYN after binding lipopolysaccharide.

Subcellular Location:

Cell membrane>Single-pass type I membrane protein. Early endosome. Cell projection>Ruffle.
Note: Upon complex formation with CD36 and TLR6, internalized through dynamin-dependent endocytosis (PubMed:20037584). Colocalizes with RFTN1 at cell membrane and then together with RFTN1 moves to endosomes, upon lipopolysaccharide stimulation.

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Tissue Specificity:

Highly expressed in placenta, spleen and peripheral blood leukocytes. Detected in monocytes, macrophages, dendritic cells and several types of T-cells.

Subunit Structure:

Belongs to the lipopolysaccharide (LPS) receptor, a multi-protein complex containing at least CD14, LY96 and TLR4. Binding to bacterial LPS leads to homodimerization. Interacts with LY96 via the extracellular domain. Interacts with MYD88 and TIRAP via their respective TIR domains (By similarity). Interacts with TICAM2. Interacts with NOX4. Interacts with CNPY3 (By similarity). Interacts with HSP90B1. The interaction with both CNPY3 and HSP90B1 is required for proper folding in the endoplasmic reticulum. Interacts with MAP3K21; this interaction leads to negative regulation of TLR4 signaling. Interacts with CD36, following CD36 stimulation by oxLDL or amyloid-beta 42, and forms a heterodimer with TLR6. The trimeric complex is internalized and triggers inflammatory response. LYN kinase activity facilitates TLR4-TLR6 heterodimerization and signal initiation. Interacts with TICAM1 in response to LPS in a WDFY1-dependent manner. Interacts with WDFY1 in response to LPS (By similarity). Interacts with SMPDL3B (By similarity). Interacts with CEACAM1; upon lipopolysaccharide stimulation, forms a complex including TLR4 and the phosphorylated form of SYK and CEACAM1, which in turn, recruits PTPN6 that dephosphorylates SYK, reducing the production of reactive oxygen species (ROS) and lysosome disruption, which in turn, reduces the activity of the inflammasome (By similarity). Interacts with RFTN1; the interaction occurs in response to lipopolysaccharide stimulation. Interacts with SCIMP; the interaction occurs in response to lipopolysaccharide stimulation and is enhanced by phosphorylation of SCIMP by LYN (By similarity). This interaction facilitates the phosphorylation of TLR4 by LYN which elicits a selective cytokine response in macrophages (By similarity).

(Microbial infection) In case of infection, interacts with uropathogenic E.coli protein TcpC.

Family&Domains:

The TIR domain mediates interaction with NOX4.

The TIR domain mediates NAD(+) hydrolase (NADase) activity. Self-association of TIR domains is required for NADase activity.

Belongs to the Toll-like receptor family.

Research Fields

· Cellular Processes > Transport and catabolism > Phagosome.   (View pathway)

· Cellular Processes > Cell growth and death > Necroptosis.   (View pathway)

· Environmental Information Processing > Signal transduction > NF-kappa B signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > HIF-1 signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > PI3K-Akt signaling pathway.   (View pathway)

· Human Diseases > Infectious diseases: Bacterial > Pathogenic Escherichia coli infection.

· Human Diseases > Infectious diseases: Bacterial > Salmonella infection.

· Human Diseases > Infectious diseases: Bacterial > Pertussis.

· Human Diseases > Infectious diseases: Bacterial > Legionellosis.

· Human Diseases > Infectious diseases: Parasitic > Leishmaniasis.

· Human Diseases > Infectious diseases: Parasitic > Chagas disease (American trypanosomiasis).

· Human Diseases > Infectious diseases: Parasitic > Malaria.

· Human Diseases > Infectious diseases: Parasitic > Toxoplasmosis.

· Human Diseases > Infectious diseases: Parasitic > Amoebiasis.

· Human Diseases > Infectious diseases: Bacterial > Tuberculosis.

· Human Diseases > Infectious diseases: Viral > Hepatitis B.

· Human Diseases > Infectious diseases: Viral > Measles.

· Human Diseases > Infectious diseases: Viral > Influenza A.

· Human Diseases > Cancers: Overview > Proteoglycans in cancer.

· Human Diseases > Immune diseases > Inflammatory bowel disease (IBD).

· Human Diseases > Immune diseases > Rheumatoid arthritis.

· Organismal Systems > Immune system > Toll-like receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > NOD-like receptor signaling pathway.   (View pathway)

References

1). SARS-CoV-2 envelope protein impairs airway epithelial barrier function and exacerbates airway inflammation via increased intracellular Cl- concentration. Signal transduction and targeted therapy, 2024 (PubMed: 38528022) [IF=39.3]

Application: WB    Species: Human    Sample: BEAS-2B cells

Fig. 3 SARS-CoV-2 envelope (E) protein induced airway inflammation via the TLR2/4-JNK-AP-1 signaling pathway. a Representative immunoblots showing the phosphorylation level of p38, ERK and JNK in BEAS-2B cells after E protein (50 μg/ml) stimulation for 12 h. GAPDH served as a loading control. b Immunofluorescence staining of lung sections showing the phosphorylation of JNK in Ad5-hACE2 transgenic mice with or without SARS-CoV-2 infection (1 × 105 PFU). Scale bar = 50 μm. c Co-immunoprecipitates and the total lysates (Input) were detected by Western Blotting using anti-TLR2 and anti-TLR4 antibody. d GST pull-down assays using GST-E protein and TLR2. TLR2 binding to GST-E protein was determined by using an anti-His antibody. e GST pull-down assays using GST-E protein and TLR4. TLR4 binding to GST-E protein was determined by using an anti-His antibody. f Representative immunoblots showing the effect of C29 (50 μM), an inhibitor of TLR2, on the phosphorylation level of JNK in BEAS-2B cells after stimulation with E protein (50 μg/ml) for 12 h. GAPDH served as a loading control. g Representative immunoblots showing the effect of Resatorvid (5 μM), an inhibitor of TLR4, on the phosphorylation level of JNK in BEAS-2B cells after stimulation with E protein (50 μg/ml) for 12 h. GAPDH served as a loading control. h Representative immunoblots showing the phosphorylation level of c-Jun in BEAS-2B cells after E protein (50 μg/ml) stimulation for 12 h. GAPDH served as a loading control. i Representative immunoblots showing the effect of SP600125 (10 μM), the selective inhibitor of JNK, on the phosphorylation level of JNK in BEAS-2B cells after stimulation with E protein (50 μg/ml) for 12 h. GAPDH served as a loading control. j Heatmap showing significant changes in mRNA expression of inflammation-related genes in BEAS-2B cells stimulated with E protein (50 μg/ml) with or without SP600125 (10 μM) for 12 h (n = 3/group). k Quantitative real-time PCR analyses showing the effect of C29 (50 μM), SP600125 (10 μM) and the selective AP-1 inhibitor T-5224 (10 μM) on the expression of tumor necrosis factor-α (TNF-α) in BEAS-2B cells stimulated with E protein (50 μg/ml) for 12 h (n = 3). l Quantitative real-time PCR analyses showing the effect of SP600125 (30 mg/kg) on the expression of TNF-α in the lung samples from mice after intratracheal instillation of E protein (100 μg/ml) for 24 h (n = 5). Data are shown as means ± S.D. **P 
2). Anti-inflammatory effect of piperine ameliorates insulin resistance in monosodium glutamate–treated obese mice. METABOLISM-CLINICAL AND EXPERIMENTAL, 2020 (PubMed: 19800637) [IF=9.8]

Application: WB    Species: Mouse    Sample: RAW264.7 cells

Figure 9. Effects of piperine on LPS-induced M1-like polarization in RAW264.7 cells. RAW264.7 cells were pretreated with piperine at 20-80 μM for 12 h and then stimulated by LPS (1 μg/ml) for 24 h. (a-c) The mRNA levels of CD11c, IL-1β and TNF-α were analyzed by qPCR. (d) The IL-1β level in cell supernatant was measured by ELISA. (e-f) The protein levels of TLR-4, CD11c and IL-1β were tested by Western Blotting. Data are expressed as mean±SD, n=3. ####p
3). Berberine-mediated up-regulation of surfactant protein D facilitates cartilage repair by modulating immune responses via the inhibition of TLR4/NF-ĸB signaling. Pharmacological Research, 2020 (PubMed: 32057894) [IF=9.3]

Application: IHC    Species: rat    Sample: knee joints

Fig. 5. Chondrocyte apoptosis and immune molecular biomarkers were inhibited by BBR binding to SP-D in vivo in OA. OA model rats (n = 5/group) were injected using 25 μl of 200 μM BBR, followed by 25 μl rhSP-D (40 μg/mL) 1 day later. Sham-operated and OA-induction groups received an injection of 50 μl PBS into the right knee joint. For rats receiving rAAV-GFP or rAAV-SP-D shRNA, injections were performed for 10 consecutive days beginning 1 day after the initial BBR injection. At 10 weeks post-surgery, animals were sacrificed and the knee joints were collected for TUNEL and IHC analysis. (A) TUNEL staining (400 ×) was used to assess apoptosis. The p-p65, TLR4, F4/80, CD68, and CD34 expression was assessed via IHC (400 × with higher 800 × magnification inset). (B) Quantification of the indicated proteins in IHC images. Each column represents the mean ± SEM (n = 5). ***P < 0.001 versus the sham-operated group; ##P < 0.01 and ###P < 0.001 versus the OA-induction group; &P < 0.05 and &&P < 0.01 versus the OA + BBR group.
4). Extracellular vesicles derived from mesenchymal stem cells alleviate neuroinflammation and mechanical allodynia in interstitial cystitis rats by inhibiting NLRP3 inflammasome activation. Journal of Neuroinflammation, 2022 (PubMed: 35387668) [IF=9.3]

Application: WB    Species: rat    Sample: spinal dorsal horn

Fig. 8 | MSC-EVs inhibit activity of TLR4/NF-κB signal pathway in SDH of IC rats. A–C Western blot analysis showing that expression level of TLR4 and phosphorylation ratio of NF‐κB (p65) were signifcantly increased in SDH of IC rats compared with normal rats, and intrathecal injection of MSC-EVs signifcantly decreased expression level of TLR4 and phosphorylation ratio of NF‐κB (p65) in SDH of IC rats
5). Arsenic trioxide ameliorates experimental autoimmune encephalomyelitis in C57BL/6 mice by inducing CD4+ T cell apoptosis. Journal of Neuroinflammation, 2020 (PubMed: 32375831) [IF=9.3]
6). Baicalin ameliorates neuroinflammation-induced depressive-like behavior through inhibition of toll-like receptor 4 expression via the PI3K/AKT/FoxO1 pathway. Journal of Neuroinflammation, 2019 (PubMed: 31068207) [IF=9.3]

Application: WB    Species: mouse    Sample: hippocampus

Fig. 3| The role of TLR4 in alleviating neuroinflammation of baicalin. Prophylactic treatment of TAK-242 or baicalin inhibited LPS-induced neuroinflammation as well as depressive-like behaviors. d The levels of TLR4 and p-FoxO1 in the hippocampus were measured by western blotting and were quantified and normalized with their respective β-actin or total FoxO1 levels (n = 4). Data were normalized to the control and presented as means ± SEM. #p < 0.05, ##p < 0.01, ###p < 0.005 vs. control; *p < 0.05, **p < 0.01, ***p < 0.005 vs. LPS. TAK, TAK-242

Application: IF/ICC    Species: mouse    Sample: BV2 cells

Fig. 7 |Baicalin regulates LPS-induced TLR4 expression by activating the PI3K/AKT/FoxO1 pathway in BV2 cells.e BV2 cells were stained with a TLR4 antibody and observed with fluorescence microscopy. Green, TLR4; blue: DAPI; scale bar, 50 μm
7). Oxyberberine, a novel gut microbiota-mediated metabolite of berberine, possesses superior anti-colitis effect: impact on intestinal epithelial barrier, gut microbiota profile and TLR4-MyD88-NF-κB pathway. PHARMACOLOGICAL RESEARCH, 2020 (PubMed: 31863867) [IF=9.3]

Application: WB    Species: Mice    Sample: colonic tissues

Fig. 6. Effect of OBB on the activation of TLR4-MyD88-NF-κB signaling pathway in DSS-induced colonic tissues. (A) Representative Western blotting images of TLR4, MyD88, cytoplasmic p65, nuclear p65, p-IκBα and IκBα. Changes in the relative protein expression levels of TLR4 (B), MyD88 (C), nuclear p65 (D), cytoplasmic p65 (E), and p-IκBα/IκBα ratio (F) were measured. Data are shown as the mean ± SEM (n = 3). # P < 0.05, ## P < 0.01 vs. Control group, * P < 0.05, ** P < 0.01 vs. DSS group.
8). Commensal cow Roseburia reduces gut-dysbiosis-induced mastitis through inhibiting bacterial translocation by producing butyrate in mice. Cell Reports, 2022 (PubMed: 36417859) [IF=8.8]
9). Dental pulp stem cell‐derived exosomes alleviate cerebral ischaemia‐reperfusion injury through suppressing inflammatory response. CELL PROLIFERATION, 2021 (PubMed: 34231932) [IF=8.5]

Application: WB    Species: Mice    Sample:

FIGURE 3 Effect of DPSC‐Exos on the expression of TLR4, MyD88, NF‐κB p65 and HMGB1 on day 7 after cerebral I/R damage. (A) The relative expression level of TLR4. (B) The relative expression level of MyD88. (C) The relative expression level of NF‐κB p65. (D) The relative expression level of nuclear HMGB1. (E) The relative expression level of cytoplasmic HMGB1. Protein samples were acquired from the ischaemic cortex and assayed by western blot. Nuclear proteins were normalized to the intensity of Histone H3, and cytoplasmic and total proteins were normalized to the intensity of GAPDH or β‐actin. Data were expressed as means ± SD (n = 3). ## P < .01 versus the sham group; *P < .05 and **P < .01 versus the I/R + PBS group. DPSC‐Exos, dental pulp stem cell‐derived exosomes; HMGB1, high‐mobility group box 1 protein; I/R, ischaemia/reperfusion; MyD88, myeloid differentiation protein 88; NF‐κB, nuclear factor‐kappa B; PBS, phosphate‐buffered saline; TLR4, toll‐like receptor‐4
10). Sodium alginate and galactooligosaccharides ameliorate metabolic disorders and alter the composition of the gut microbiota in mice with high-fat diet-induced obesity. International Journal of Biological Macromolecules, 2022 (PubMed: 35718141) [IF=8.2]
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