Human LILRB4/CD85k/ILT3 Antibody

R&D Systems | Catalog # AF2425

R&D Systems

Key Product Details

Species Reactivity

Validated:

Human

Cited:

Human

Applications

Validated:

Western Blot, Flow Cytometry, CyTOF-ready

Cited:

Neutralization

Label

Unconjugated

Antibody Source

Polyclonal Goat IgG
View all LILRB4/CD85k/ILT3 Primary Antibodies »

Product Specifications

Immunogen

Mouse myeloma cell line NS0-derived recombinant human LILRB4/CD85k/ILT3
Pro17-His257
Accession # ABM86208

Specificity

Detects human LILRB4/CD85k/ILT3 in direct ELISAs and Western blots. In direct ELISAs, approximately 15% cross‑reactivity with recombinant human (rh) ILT5 is observed and 10% cross-reactivity with rhILT4 and rhILT2 is observed.

Clonality

Polyclonal

Host

Goat

Isotype

IgG

Scientific Data Images for Human LILRB4/CD85k/ILT3 Antibody

Detection of LILRB4/CD85k/ILT3 by Western Blot

Detection of LILRB4/CD85k/ILT3 by Western Blot

Galectin-9 interacts with Vamp-3 in DCs. A moDCs were lysed and whole-cell extracts incubated with either anti-galectin-9 specific antibody or an isotype control (goat IgG). All interacting proteins were isolated and identified by mass-spectrometry. Volcano plot depicts differentially enriched galectin-9 (right) and isotype (left) interacting proteins. B moDCs were prepped as per (A) but immunoprecipitated (IP) complexes were resolved and probed with galectin-9 and Vamp-3 specific antibodies. Graph shows quantification of Vamp-3 content in each sample using ImageJ. Data shown is representative of 5 individual experiments. C THP-1 cells were transfected with empty vector or plasmid containing 3 × FLAG_Vamp-3, lysed and whole-cell extracts incubated with either anti-galectin-9 specific antibody or an isotype control (goat IgG). IP complexes were resolved and probed with galectin-9 and Vamp-3 specific antibodies. Immunoblot shown is representative of four independent experiments. D NT or gal9 siRNA transfected moDCs were treated with LPS or DMSO as negative control for 6 h prior to being lysed and whole lysates probed against the specified proteins. Tubulin was used as loading control. Immunoblot shown is representative of three independent experiments. Graph shows quantification of Vamp-3 content in each sample using ImageJ. E. Quantification of Vamp-3 and SNAP23 levels shown in (D). Graphs depict Vamp-3 and SNAP23 levels in galectin-9-depleted moDCs normalised to tubulin and made relative to the corresponding untreated NT siRNA sample. Graph shows mean ± SD of three independent donors. **p < 0.005 Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37755527), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of LILRB4/CD85k/ILT3 by Immunocytochemistry/ Immunofluorescence

Detection of LILRB4/CD85k/ILT3 by Immunocytochemistry/ Immunofluorescence

Galectin-9 regulates Vamp-3 intracellular localisation. A NT and gal9 siRNA transfected moDCs were incubated with LPS for 4 h prior to being fixed in methanol and stained for Vamp-3, galectin-9 and DAPI for nuclear localisation. Representative images for each treatment are shown. Graphs: fluorescent cross-sections as indicated depicting Vamp-3 intensity across the cell. Scale bar: 10 µm. B Line scan quantification of cells shown in (A). Data represent mean Vamp-3 intensity for the specified distance from the nuclei ± SEM of one representative donor out of three. Fifteen cells were analysed per condition. Two-way ANOVA was performed for statistical analysis. C Representative images of DCs treated as in (A), fixed in 4% PFA and stained against galectin-9, Vamp-3, PDI, GM130, EEA1 and DAPI for nuclear localisation. Graph depicts Vamp-3 integrated density values for three independent donors ± SEM. Fifteen cells were analysed per donor. Unpaired t-test was conducted between NT and gal9 siRNA samples. Please note that PFA-fixed cells also show Vamp-3 nuclear staining as a result of the fixation process. n.s. p > 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37755527), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of LILRB4/CD85k/ILT3 by Immunocytochemistry/ Immunofluorescence

Detection of LILRB4/CD85k/ILT3 by Immunocytochemistry/ Immunofluorescence

Galectin-9-depleted moDCs eliminate intracellular cytokines via lysosomes. A, B Day 6 NT and gal9 siRNA-transfected moDCs were treated with LPS (1 µg/ml) alone or with Bafilomycin (200 µM) for 6 h after which cells were harvested and intracellular levels of TNF alpha determined by flow cytometry. Graph represents the % of TNF alpha positive cells (A) or the amount of TNF alpha present intracellularly represented by the mean fluorescence TNF alpha intensity (B). Graphs show mean value ± SEM of three independent donors. Paired students t-test was conducted between NT and gal9 siRNA-transfected cells. C Representative images from NT and gal9 siRNA moDCs treated as per (A). Scale bar: 10 µm. D Confocal microscopy quantification of the number of TNF alpha -positive cells (%) (left) and the integrated TNF alpha intensity of such cells (right) in NT and gal9 siRNA DCs treated with LPS and Bafilomycin. Data represent mean ± SEM of two independent donors. Unpaired Student's t-test was conducted between NT and gal9 siRNA-transfected cells. *p < 0.05; **p < 0.005 Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37755527), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of LILRB4/CD85k/ILT3 by Immunocytochemistry/ Immunofluorescence

Detection of LILRB4/CD85k/ILT3 by Immunocytochemistry/ Immunofluorescence

Post-Golgi cytokine trafficking is impaired upon galectin-9 depletion. A Scheme depicting the experimental design based on the RUSH system. In the absence of biotin (left panel), the TNF alpha _GFP is trapped at the ER by the lumenal Str-KDEL hook and no cytokine trafficking post-ER can occur. When biotin is added (right panel), biotin outcompetes the interaction with streptavidin, allowing TNF alpha _GFP to freely traffic to the Golgi and the endosomal compartments. B NT and gal9 siRNA transfected moDCs were transfected with a GFP-labelled TNF alpha RUSH construct and treated with biotin to induce GFP-TNF alpha trafficking. Images represent snapshots of live-cell imaging GFP-TNF alpha at the indicated time points. Scale bar: 10 µm. C Quantification of images shown in (B). Data represent mean intensity ± SEM from two independent donors. D moDCs treated as per (B) were incubated with biotin for 30 min prior to being fixed and stained for GM130 as Golgi marker, galectin-9 and DAPI for nuclear localisation. Representative images are shown. Scale bar: 10 µm. E Representative line scans of cells shown in (D) depicting GM130 (yellow) and TNF alpha (in red) intensity across the cell. F. Mander's correlation coefficient was calculated for images shown in (D). Paired Student’s t-test was conducted between NT and gal9 siRNA transfected moDCs. Data represent mean correlation coefficient ± SEM of two independent donors. Fifteen cells were analysed for each condition per donor. ***p < 0.001 Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37755527), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of LILRB4/CD85k/ILT3 by Immunocytochemistry/ Immunofluorescence

Detection of LILRB4/CD85k/ILT3 by Immunocytochemistry/ Immunofluorescence

Cytokines accumulate intracellularly upon galectin-9 depletion. A Day 5 NT and gal9 siRNA transfected moDCs were treated for 6 h with 1 µg/ml LPS alone or in combination with the Golgi inhibitors Brefeldin A (BFA, 10 µg/ml) and Monensin (Mon, 2 µM) prior to being harvested and the intracellular levels of TNF alpha analysed by flow cytometry. Dotted line shows TNF alpha levels in untreated cells. Dark grey depicts NT siRNA DCs treated with LPS (1 µg/ml) and light grey NT siRNA cells treated with LPS + the Golgi inhibitors. Green depicts gal9 siRNA DCs treated with LPS and magenta shows gal9 siRNA treated with LPS + Golgi inhibitors. B Quantification and statistical analysis of experiments depicted in (A). Results show the mean value ± SEM for four independent donors. Unpaired students t-test was conducted between NT and gal9 siRNA-transfected cells. C Representative images from results shown in (A) of cells treated with LPS only and stained against TNF alpha, galectin-9 and DAPI for nuclear localisation. Scale bar: 10 µm. D, E Confocal microscopy quantification of the number of TNF alpha positive cells (%) (D) and the integrated TNF alpha intensity of such cells (E) in NT and gal9 siRNA DCs treated with LPS. Data are representative of four independent donors. Unpaired Student's t-test was conducted between NT and gal9 siRNA-transfected cells. **p < 0.005, *p < 0.05 Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37755527), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Human LILRB4/CD85k/ILT3 Antibody

Application
Recommended Usage

CyTOF-ready

Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.

Flow Cytometry

2.5 µg/106 cells
Sample: Human whole blood monocytes

Western Blot

0.1 µg/mL
Sample: Recombinant Human LILRB4/CD85k/ILT3

Reviewed Applications

Read 2 reviews rated 4 using AF2425 in the following applications:

Flow Cytometry Panel Builder

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Advanced Features

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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Antigen Affinity-purified

Reconstitution

Reconstitute at 0.2 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: LILRB4/CD85k/ILT3

ILT3, also known as leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4) and leukocyte immunoglobulin-like receptor 5 (LIR-5), is a type I membrane protein that functions as a receptor for class I MHC antigens. It contains cytoplasmic ITIM motifs and is involved in the down-regulation of immune responses.

Long Name

Leukocyte Immunoglobulin-like Receptor, Subfamily B (with TM and ITIM Domains), Member 4

Alternate Names

CD85k, HM18, ILT3, LIR5

Entrez Gene IDs

11006 (Human); 14728 (Mouse); 107128256 (Cynomolgus Monkey)

Gene Symbol

LILRB4

UniProt

ABM86208

Additional LILRB4/CD85k/ILT3 Products

Product Documents for Human LILRB4/CD85k/ILT3 Antibody

Certificate of Analysis

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Product Specific Notices for Human LILRB4/CD85k/ILT3 Antibody

For research use only

Citations for Human LILRB4/CD85k/ILT3 Antibody

Customer Reviews for Human LILRB4/CD85k/ILT3 Antibody (2)

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  • Human LILRB4/CD85k/ILT3 Antibody
    Name: Anonymous
    Application: ELISA
    Sample Tested: Serum
    Species: Human
    Verified Customer | Posted 11/29/2022
    Human LILRB4/CD85k/ILT3 Antibody AF2425
  • Human LILRB4/CD85k/ILT3 Antibody
    Name: Anonymous
    Application: ELISA
    Sample Tested: Serum and Plasma and AML-193 human acute monocytic leukemia cell line
    Species: Human
    Verified Customer | Posted 07/07/2020
    Human LILRB4/CD85k/ILT3 Antibody AF2425

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