Anti il 23r

Manufactured by R&D Systems

Sourced in United States, Italy

Anti-IL-23R is a monoclonal antibody that binds to the interleukin-23 receptor (IL-23R). It can be used in research applications to study the role of IL-23 signaling.

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APC-anti-IL-23R Anti-IL-23R Ab IL-23R

5 protocols using anti il 23r

Molecular Profiling of Corneal Inflammation

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Mouse corneal samples were lysed with RIPA buffer. The lysates were centrifuged to obtain supernatant. Protein concentration was determined by BCA assay. For Western blot analysis, the protein samples were separated by SDS-PAGE and electrically transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5% milk and subsequently incubated with primary and secondary antibodies. Signals were visualized using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Pittsburgh, PA). β-actin was used as the loading control. Quantification of protein levels was based on the densitometry of blots by using the software Carestream MI SE (Informer Technologies, Rochester, NY). The antibodies used included: anti-IL-23, anti-IL-23R, anti-IL-17A, anti-IL-17RC, anti-MCPIP-1, anti-Osteoprotegerin (R&D), and anti-β-actin (A1978; Sigma-Aldrich). Enzyme-linked immunosorbent assay (IL-23; R&D) and protein array (Proteome Profiler Array Mouse XL Cytokine Array Kit; R&D) were performed following manufacturer’s protocols.

Me R., Gao N., Dai C, & Yu F.S. (2019). Interleukin-17 Promotes Pseudomonas aeruginosa Keratitis in C57BL/6 Mouse Corneas. Journal of immunology (Baltimore, Md. : 1950), 204(1), 169-179.

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Esophageal Squamous Cell Carcinoma Cell Lines

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The human ESCC cell lines TE-1 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and TE-10, KYSE-150, and ECA-109 cells were a gift from Dr. Ye Hua of the Clinical Medicine College at Jiangsu University. The human esophageal epithelial cell line, Het-1A, was purchased from Jennio Biological Technology (Guangzhou, China). Cells were respectively maintained in RPMI 1640 or DMEM (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (HyClone, Logan, UT, USA) and cultured at 37 °C in 5% CO₂. IL-23, anti-IL-23, and anti-IL-23R were purchased from R&D Systems (Minneapolis, MN, USA), and WHI-P154, DAPT, and ICG-001 were purchased from Selleck Chemicals Inc. (Houston, TX, USA). For RNA interference, cells were transfected with siRNA oligos (Gene Pharma, Shanghai, China) against human β-catenin and Notch1 using Lipofectamine 2000 (Invitrogen, San Diego, CA, USA). The following siRNA sequences (GenePharma, Shanghai, China) were used: siRNA-β-catenin: sense, 5′-GUCCUGUAUGAGUGGGAACTT-3′; antisense, 5′-GUUCCCACUCAUACAGGACTT-3′. siRNA-Notch1: sense, 5′-GGGCUAACAAAGAUAUGCATT-3′; antisense, 5′-UGCAUAUCUUUGUUAGCCCTT-3′. Negative controls using non-transfected cells and empty vector-transfected cells were performed in parallel. The colony formation assay and soft agar assay were described previously [22 (link)].

Zhou Y., Su Y., Zhu H., Wang X., Li X., Dai C., Xu C., Zheng T., Mao C, & Chen D. (2018). Interleukin-23 receptor signaling mediates cancer dormancy and radioresistance in human esophageal squamous carcinoma cells via the Wnt/Notch pathway. Journal of Molecular Medicine (Berlin, Germany), 97(2), 177-188.

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Dissecting Th17 Cell Subsets in Corneal Inflammation

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Draining lymph node cells from DED mice were harvested and their CD4⁺ T cells were enriched via negative selection with CD4⁺ T cell isolation kit (Miltenyi Biotec Inc.). Thereafter, Th17 (IL-17A⁺IFN-γ⁻), Th17/1 (IL-17A⁺IFN-γ⁺), and Th1 (IL-17A⁻IFN-γ⁺) subsets were further sorted using IL-17A and IFN-γ cytokine secretion assay kits (Miltenyi Biotec Inc.) and a BD FACSAria™ sorter (BD Biosciences). Each of these subsets (2 × 10⁴ cells) was subsequently injected intravenously into naïve B6.Rag1 KO mice, which were then placed in the CEC for 5 days. In the Ab treatment studies, sorted Th17 cells were injected intravenously into naïve B6.Rag1 KO mice, which were then placed in the CEC for 5 days. These recipient mice were injected intraperitoneally with 200 μg of anti-IL-12 (R & D Systems), 100 μg of anti-IL-23R (R & D Systems), 100 μg of anti-IL-12p40 Abs (R & D Systems), or 100 μg of isotype IgG (R & D Systems) one day before and one day after the cell transfer. Disease severity was evaluated using corneal fluorescein staining (CFS) described above.

Chen Y., Chauhan S.K., Shao C., Omoto M., Inomata T, & Dana R. (2017). Interferon-γ-expressing Th17 cells are required for development of severe ocular surface autoimmunity. Journal of immunology (Baltimore, Md. : 1950), 199(3), 1163-1169.

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Neutralization and Inhibition Experiments of V-γ9V-δ2 T Cells

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For neutralization experiments, PBMCs from BCG-vaccinated monkeys were cultured with media, HMBPP, HMBPP+IL-23, HMBPP+IL-23+anti-IFN-γ (1 or 5 μg/mL), HMBPP+IL-23+anti-IL-4 (5 μg/mL), HMBPP+IL-23+ anti-TGF-β (5 μg/mL) or HMBPP+IL-23+ anti-IL-17 (5 μg/mL), anti-IL-23R (5 μg/mL, R&D) or mouse IgG (5 μg/mL), with cytokine and Ab supplemented at day 0, 3, 5. At day 7, the cells were collected and stained. The following neutralizing Abs were used: anti-IFN-γ (clone MD-1; eBioscience), and anti-TGF-β (clone 9016; R&D systems), anti-IL-17 (Clone 133617, R&D Systems,), anti-IL-23R (clone 3D7, R&D Systems). Other primary and secondary mAbs or mouse isotype control IgG were listed previously [40 (link)].
For PKC inhibition experiment, PBMCs from BCG-vaccinated monkeys were cultured with media alone, HMBPP, HMBPP+IL-2, or HMBPP+IL-23; PKC inhibitors Gö6976 (500 nM, Calbiochem, San Diego, CA) and rottlerin (5 μM, Calbiochem) were added at days 3 and 5, respectively, as previously described [45 (link)]. At day 7, the cells were collected and stained.

Shen H., Wang Y., Chen C.Y., Frencher J., Huang D., Yang E., Ryan-Payseur B, & Chen Z.W. (2015). Th17-related cytokines contribute to recall-like expansion/effector function of HMBPP-specific Vγ2Vδ2 T cells after Mycobacterium tuberculosis infection or vaccination. European journal of immunology, 45(2), 442-451.

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Immunophenotyping of T Helper Cells

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Mononuclear cells were treated for 30 min at room temperature with commercially available FITC-, PE-, or PE-Cy5-conjugated monoclonal antibody (mAb): anti-CD4 (Becton-Dickinson Biosciences, Italy), anti-IL23R (R&D Systems Inc., Minneapolis, MN, USA), and anti-IL-17 (R&D Systems); anti-CD161 (Becton-Dickinson Biosciences, Italy). FITC-, PE-, or PE-Cy5-conjugated isotype-matched mouse mAb was used to set the fluorescence background (IgG1 and IgG2a, BD Pharmigen). Appropriate isotype controls and fluorescence minus one (FMO) controls were used to assign gates. Expression of the cytoplasmic cytokine was evaluated as previously described [15] . CD4+ T cells were gated with two different approaches: physical characteristic of cells and expression of CD4 in the area of lymphocytes to identify the T helper cells. Fluorescence-activated cell sorter (FACS) analysis was assessed as previously described, briefly, 5 × 105 cells were acquired on FACSCalibur 6 analyzer (Becton-Dickinson) and data processed by DIVA software program (Becton-Dickinson Biosciences, Italy). To evaluate whether differences between peaks were statistically significant with respect to controls, the Kolmogorov-Smirnov test was used.

Fiocco U., Stramare R., Martini V., Coran A., Caso F., Costa L., Felicetti M., Rizzo G., Tonietto M., Scanu A., Oliviero F., Raffeiner B., Vezzù M., Lunardi F., Scarpa R., Sacerdoti D., Rubaltelli L., Punzi L., Doria A, & Grisan E. (2017). Quantitative imaging by pixel-based contrast-enhanced ultrasound reveals a linear relationship between synovial vascular perfusion and the recruitment of pathogenic IL-17A-F⁺IL-23⁺ CD161⁺ CD4⁺ T helper cells in psoriatic arthritis joints. Clinical rheumatology, 36(2).

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