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Ab120952

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Ab120952 is a recombinant monoclonal antibody that recognizes human IL-6. It is intended for use in research applications.

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Lab products found in correlation

Ab144824, by Abcam (2 mentions) Il 13, by BioLegend (2 mentions) L2630, by BioLegend (2 mentions) Ab120234, by Abcam (1 mentions) Ab141406, by Abcam (1 mentions) Hk 2 cells, by National Collection of Authenticated Cell Cultures (1 mentions) Ab17147, by Abcam (1 mentions) Anti β actin actb antibody, by Merck Group (1 mentions) Mkn45, by RIKEN Cell Bank (1 mentions) Mkn74, by RIKEN Cell Bank (1 mentions) Hgc27, by RIKEN Cell Bank (1 mentions) Nugc4, by RIKEN Cell Bank (1 mentions) Rpmi 1640, by Nacalai Tesque (1 mentions) Pe anti pd l1 antibody, by BioLegend (1 mentions) Recombinant mouse il 17a 210 17, by Thermo Fisher Scientific (1 mentions)

6 protocols using ab120952

1

Modulation of RASF Activation by Cytokines

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RASFs were seeded into 24-well plates (density 2–4 × 104/well) Then cells were cultrued for 12, 24, 48 h in the presence of 0, 1, 10, 50 ng/ml of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), or lipopolysaccharide (LPS) respectively, or combined with 10μmol/l of PDTC (an inhibitor of nuclear transcription factor kappa B (NF-кB), Abcam, ab141406), PD98059 (an inhibitor of extracellular regulated protein kinases (ERK-1/2), Abcam, ab120234), or Static (an inhibitor of signal transducers and activators of transcription (STAT3), Abcam, ab120952), respectively. For the siRNA blocking assay, 0, 1, 10, 50 ng/ml of TNF-α with or without 100 nmol/l siRNA-E2F2 were added to seeded RASFs for 24 h (see below).
Zhang R., Wang L., Pan J.H, & Han J. (2018). A critical role of E2F transcription factor 2 in proinflammatory cytokines-dependent proliferation and invasiveness of fibroblast-like synoviocytes in rheumatoid Arthritis. Scientific Reports, 8, 2623.
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2

Murine Bone Marrow-Derived Macrophage Culture

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BMDMs were obtained by in vitro differentiation of primary femur and tibia bone marrow cells using an established protocol(Davies and Gordon, 2005 ; Healy et al., 2020 ). In brief, femurs and tibias from the indicated mouse lines mice were dissected, cleaned, dis-infected in 70% ethanol, and washed with fully supplemented RPMI 1640 medium. Red blood cells were removed by ammonium-chloride potassium lysis buffer and subsequent centrifugation. Remaining BM cells were then cultured at a density of 3.5 × 106 cells/10 cm Petri dish in fully supplemented RPMI with 30% (vol/vol) L929 cell-conditioned medium. Cells were matured to phenotypic macrophages over 6–7 days as confirmed by >98% expression of F4/80 and CD68. Non-adherent cells were washed away with PBS while adherent cells were recovered by gentle pipetting in PBS with 1 mM EDTA. Polarization of BMDMs was carried out by incubation with LPS (100 ng/mL; Sigma-Aldrich L2630) and IFN-γ (50 ng/mL; BioLegend 575304) or with IL-4 (10 ng/mL; BioLegend 574304) and IL-13 (10 ng/mL; BioLegend cat.575904) up to 24 hours. In some experiments, BMDMs were treated with the STAT3 inhibitor, Stattic (Abcam, ab120952), or the NF-κB inhibitor, JSH23 (Abcam, ab144824).
Mantsounga C.S., Lee C., Neverson J., Sharma S., Healy A., Berus J.M., Parry C., Ceneri N.M., López-Giráldez F., Chun H.J., Lu Q., Sellke F., Choudhary G, & Morrison A.R. (2022). Macrophage IL-1β promotes arteriogenesis by autocrine STAT3- and NF-κB-mediated transcription of pro-angiogenic VEGF-A. Cell reports, 38(5), 110309.
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3

CCL5 Regulation of HK-2 Cells

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HK-2 cells were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China) and cultured in DMEM-F12 supplemented with 10% fetal bovine serum at 37°C with 5% CO2. To investigate the effect of CCL5, HK-2 cells were exposed to 0.5 or 1 μg/ml CCL5 (sc-4,637, Santa Cruz) for 24 h. To verify the effect of STAT3, STAT3 inhibitor stattic (10 μM, ab120952, Abcam) was applied. To verify the effect of DNMT1, DNMT1 inhibitor 5-Aza (10 μM) was applied. Cells were harvested for western blotting analysis.
Liu Q., Li S., Yu L., Yin X., Liu X., Ye J, & Lu G. (2022). CCL5 Suppresses Klotho Expression via p-STAT3/DNA Methyltransferase1-Mediated Promoter Hypermethylation. Frontiers in Physiology, 13, 856088.
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4

Murine Bone Marrow-Derived Macrophage Culture

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BMDMs were obtained by in vitro differentiation of primary femur and tibia bone marrow cells using an established protocol(Davies and Gordon, 2005 ; Healy et al., 2020 ). In brief, femurs and tibias from the indicated mouse lines mice were dissected, cleaned, dis-infected in 70% ethanol, and washed with fully supplemented RPMI 1640 medium. Red blood cells were removed by ammonium-chloride potassium lysis buffer and subsequent centrifugation. Remaining BM cells were then cultured at a density of 3.5 × 106 cells/10 cm Petri dish in fully supplemented RPMI with 30% (vol/vol) L929 cell-conditioned medium. Cells were matured to phenotypic macrophages over 6–7 days as confirmed by >98% expression of F4/80 and CD68. Non-adherent cells were washed away with PBS while adherent cells were recovered by gentle pipetting in PBS with 1 mM EDTA. Polarization of BMDMs was carried out by incubation with LPS (100 ng/mL; Sigma-Aldrich L2630) and IFN-γ (50 ng/mL; BioLegend 575304) or with IL-4 (10 ng/mL; BioLegend 574304) and IL-13 (10 ng/mL; BioLegend cat.575904) up to 24 hours. In some experiments, BMDMs were treated with the STAT3 inhibitor, Stattic (Abcam, ab120952), or the NF-κB inhibitor, JSH23 (Abcam, ab144824).
Mantsounga C.S., Lee C., Neverson J., Sharma S., Healy A., Berus J.M., Parry C., Ceneri N.M., López-Giráldez F., Chun H.J., Lu Q., Sellke F., Choudhary G, & Morrison A.R. (2022). Macrophage IL-1β promotes arteriogenesis by autocrine STAT3- and NF-κB-mediated transcription of pro-angiogenic VEGF-A. Cell reports, 38(5), 110309.
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5

Gastric Cancer Cell Line Characterization

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The GC cell lines MKN7, MKN45, MKN74, HGC27, and NUGC4 were purchased from the Riken Cell Bank. These cells were cultured in RPMI‐1640 (Nacalai Tesque) supplemented with 100 μg/mL of streptomycin, 100 U/mL of penicillin, and 10% fetal bovine serum (FBS). Cells were cultured at 37°C in a 5% CO2 incubator. The rabbit polyclonal anti‐ANO9 antibody used in the immunohistochemical (IHC) analysis and western blotting was purchased from Abcam (ab140087). The rabbit polyclonal anti‐PD‐L2 antibody used in the IHC analysis was purchased from ProteinTech (18251‐1‐1AP). The mouse monoclonal anti‐PD‐L1 antibody was purchased from Cell Signaling Technology (#13684), the mouse monoclonal anti‐CD8 antibody was purchased from Abcam (ab17147) and the mouse monoclonal anti‐β‐actin (ACTB) antibody was purchased from Sigma‐Aldrich. Horseradish peroxidase (HRP)‐conjugated anti‐rabbit and mouse secondary antibodies were obtained from Cell Signaling Technology. We obtained PE anti‐PD‐L1 antibody (#329706) and APC anti‐PD‐L2 antibody (#329608) from Biolegends and recombinant human PD‐1 (PE) from Abcam (ab246145). We purchased IFNα from EnoGene Biotechnology (E6L00101) and STAT3 inhibitor from Abcam (ab120952).
Katsurahara K., Shiozaki A., Kosuga T., Shimizu H., Kudou M., Arita T., Konishi H., Komatsu S., Kubota T., Fujiwara H., Okamoto K., Kishimoto M., Konishi E, & Otsuji E. (2021). ANO9 regulates PD‐L2 expression and binding ability to PD‐1 in gastric cancer. Cancer Science, 112(3), 1026-1037.
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6

IL-17A Signaling Pathway Inhibition

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Recombinant human IL-17A (AF-200-17) and recombinant mouse IL-17A (210-17) were obtained from Pepro Tech (USA) and reconstituted in RPMI-1640 medium at appropriate concentrations. Sttatic STAT3 inhibitor (ab120952) was purchased from Abcam and reconstituted in DMSO to 50 mM.
Gunjigake K., Kinoshita J., Yamaguchi T., Saito H., Fujimori D., Horiike T., Harada S., Tajima H., Ninomiya I., Ohta T, & Fushida S. (2020). Interleukin-17A derived from mast cells contributes to fibrosis in gastric cancer with peritoneal dissemination. Gastric Cancer, 24(1), 31-44.
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