The human IL-23p19 ELISA kit was obtained from Abcam; CXCL9, CXCL10, and CXCL11Quantikine ELISA kitswere purchased fromR&D Systems. Human phosphorylated gp130was measured in cell lysates (25 and 12.5 mg per well) with a sandwich ELISA (R&D Systems) according to the manufacturer’s instructions. The binding of p19 and vIL-6 to gp130 was measured through themodification of a reportedmethod (27 (link)). Recombinant human soluble gp130 (R&D Systems) was immobilized on ELISA plate wells (4HBX, Immulon) at 5 μg/ml in phosphate-buffered saline (PBS).After blocking (SuperBlock, Life Technologies), maltose-binding protein (MBP) fused to vIL-6 (MBP–vIL-6) (27 (link)) or p19 (IL-23A, Abnova) was applied at various concentrations in PBS, 1% BSA, and the mixtures were incubated for 4 hours at room temperature. Bound proteinwas detectedwith polyclonal rabbit IgG against vIL-6 (27 (link)) or goat IgGagainst p19 (AF1716,R&DSystems; both at 1 μg/ml), followed by horseradish peroxidase (HRP)–conjugated donkey anti-rabbit IgG (NA934V,GEHealthcare Life Sciences) or rabbit anti-goat IgG (401504, Calbiochem) at a 1:3000 dilution in PBS, 0.05% Tween 20. Reactions were visualized with tetramethoxybenzene peroxidase substrate, followed by 2NH2SO4. Plates were read at 450 nm with a microplate reader.
Cxcl9
Manufactured by Abcam
Sourced in United States
CXCL9 is a chemokine that plays a role in the recruitment and activation of T cells. It is involved in the inflammatory response and immune system processes.
Automatically generated - may contain errors
Lab products found in correlation
Na934v, by GE Healthcare (1 mentions)
Superblock, by Thermo Fisher Scientific (1 mentions)
Af1716, by R&D Systems (1 mentions)
Sandwich elisa, by R&D Systems (1 mentions)
Ctla4, by Abcam (1 mentions)
Pd l1, by Abcam (1 mentions)
Genegnome hr system, by Syngene (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Ecl plu, by Cytiva (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
3 protocols using cxcl9
1
Quantifying gp130 Binding Interactions
2
Breast Cancer Biomarker Expression Analysis
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Breast cancer tissue samples were collected from a series of 115 patients with early-stage breast cancer diagnosed in the Cancer Hospital of SUMC in 2016-2017. The study and related procedures were approved by the Ethical Review Board of the hospital as indicated. Immunohistochemistry was performed as described previously (20 (link)). Briefly, slides were deparaffinized and rehydrated, and antigen retrieval was performed. Subsequently, endogenous peroxidase activity was blocked with H2O2, and the sections were incubated with primary antibodies against CXCL9 (Abcam, Danvers, MA, USA, 1:200), PD1 (Abcam, Danvers, MA, USA, 1:200), PD-L1 (Abcam, Danvers, MA, USA, 1:200), LAG3 (Abcam, Danvers, MA, USA, 1:200) and CTLA4 (Abcam, Danvers, MA, USA, 1:200) for 1 hour at room temperature (RT). After that, the tissue sections were incubated for 30 min at RT with HRP-conjugated secondary antibodies and stained with DAB. Two independent pathologists evaluated the staining score according to a method described previously (21 (link)).
3
Immunoblotting of Chemokines in Cancer Cells
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MNK28 cells, SGC7901 cells, MNK28 cells transfected with GV268-miR-588, and SGC7901 cells transfected with GV268-miR-588 were harvested. For immunoblotting, cells were lysed by radioimmunoprecipitation assay (RIPA) buffer (P0013C, Beyotime) and centrifugated at 10,000 × g for 10 minutes. Extracted proteins were then resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Protein blots were incubated with appropriate antibodies, and protein bands were visualized by enhanced chemiluminescence (ECL) or ECLPLUS (Amersham, Piscataway, NJ). The antibodies used were CXCL5 (D263012-0025, BBI), CXCL9 (ab202961, Abcam), CXCL10 (D220389-0025, BBI), and antitubulin (#T8203, Sigma-Aldrich, St. Louis, MO). The GeneGnome HR system (Syngene, Cambridge, UK) was used to scan blots.
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