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Il 6 blocking antibody

Manufactured by R&D Systems
Sourced in United States

The IL-6 blocking antibody is a laboratory research tool designed to inhibit the activity of the cytokine interleukin-6 (IL-6). It functions by binding to IL-6, thereby preventing its interaction with cellular receptors. This antibody is intended for use in in vitro and ex vivo research applications involving the study of IL-6 signaling pathways and their modulators.

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4 protocols using il 6 blocking antibody

1

In vitro invasion assay with FABP4

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An in vitro invasion assay was performed in triplicate using Growth Factor Reduced BD BioCoat Matrigel Invasion Chambers (BD Biosciences) according to the manufacturer’s instructions. Briefly, 3 × 104 cells were seeded in the upper chamber with conditioned medium from PrSC treated with or without 100ng ml-1 rFABP4, and the presence or absence of 10  μg ml-1 of IL-8 blocking antibody, IL-6 blocking antibody, control goat IgG, or control mouse IgG (R&D Systems, Minneapolis, MN, USA). In the siRNA experiments, cells were treated with 50 nM FABP4 siRNAs for 24 hours before being seeded in the chambers. Subsequently, 20% FBS DMEM was placed in the lower chamber, followed by incubation for 24 hours. In some experiments, 1 × 104 PrSC were seeded in the lower chamber with optimal medium. Sera from mice were collected, clarified by filtration (SLGV004SL; Millipore, Billerica, MA, USA), and used for ex vivo cell invasion assays. Briefly, 3 × 104 cells were seeded in the upper chamber with medium containing 5% FBS or 5% mouse serum with or without 10  μg ml-1 of IL-8 blocking antibody or 30 μM of BMS309403. DMEM with 20% FBS was placed in the lower chamber. Then, the non-invading cells in the upper chamber were removed and the membranes were stained with a Diff-Quik cell-staining kit (Sysmex, Kobe, Japan) to count the invading cells. The experiments were performed twice each in triplicate.
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2

Myeloma Cell-Stromal Cell Coculture Assay

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Cocultures were seeded into 384 well plates (Corning) with the Multidrop Combi (Thermo Scientific). 800 stromal cells were seeded and incubated overnight. 17 hours later, 700 myeloma cells were added on top of the stromal cells. On the third day, 24 hours after the addition of myeloma cells, cocultures were treated with tofacitinib (LC Laboratories), ruxolitinib (Selleck Chemicals), JAK3i,20 (link) or IL-6 blocking antibody (R&D Systems). For drug combination studies, on the fourth day, melphalan (Sigma Aldrich), carfilzomib (Selleck Chemicals), or venetoclax (Selleck Chemicals) were additionally added to cocultures. On the fifth day, myeloma cell viability was detected with the addition of luciferin (Gold Biotechnology) and read for luminescence on Glomax Explorer plate reader (Promega) as previously described.21 (link) For monoculture studies cell viability was measured using CellTiter-Glo reagent (Promega). All measurements were performed in quadruplicate. All viability data are reported as normalized to dimethyl sulfoxide (DMSO)-treated cell line in monoculture.
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3

Immunoblotting and ChIP Assays for Cell Signaling Analysis

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The following antibodies were used at the indicated dilutions for immunoblotting: pJNK (#9251, 1:1000), p-p38 (#9211, 1:1000), pERK1/2 (#9101, 1:1000) and pSTAT3 antibodies (#9131, 1:1000), obtained from Cell Signaling; JNK (C-17, 1:1000), p38 (H-147, 1:1000), ERK1/2 (K-23, 1:1000), TRAF6 (H-274, 1:1000), anti-ubiquitin (P4D1, 1:1000), RAN (C-20, 1:1000), STAT3 (C-20, 1:1000), c-Myc (9E10, 1:5,000) and tubulin antibodies (10D8, 1:2000), obtained from Santa Cruz Biotech; and anti-K63 ubiquitin (HWA4C4, 1:750) and anti-Ubc13 antibodies (#371100, 1:1000), obtained from Enzo and Invitrogen, respectively. TRAF6 antibody (H-274) was also used for immunoprecipitation (1 g/sample). For ChIPs, the following antibodies were used at 2 g/sample: STAT3 (C-20) and c-Ets-1 antibodies (C-20), obtained from Santa Cruz Biotech; H3K4me3 (CS200580) and total H3 antibodies (04-928), obtained from Millipore; and Set1 antibody (A300-289A) from Bethyl Laboratories. For flow cytometry, fluorescently labeled antibodies to CD11b (M1/70, FITC-labeled), F4/80 (BM8, PerCP Cy5.5-labeled), M-CSFR (AFS98, APC-labeled) and RANK (R12-31, PE-labeled) from eBioscience were used at a 1:100 dilution. Recombinant murine RANKL, IL-6, IL-10 and IL-6 blocking antibody were purchased from R&D Systems.
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4

Immunoblotting and ChIP Assays for Cell Signaling Analysis

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The following antibodies were used at the indicated dilutions for immunoblotting: pJNK (#9251, 1:1000), p-p38 (#9211, 1:1000), pERK1/2 (#9101, 1:1000) and pSTAT3 antibodies (#9131, 1:1000), obtained from Cell Signaling; JNK (C-17, 1:1000), p38 (H-147, 1:1000), ERK1/2 (K-23, 1:1000), TRAF6 (H-274, 1:1000), anti-ubiquitin (P4D1, 1:1000), RAN (C-20, 1:1000), STAT3 (C-20, 1:1000), c-Myc (9E10, 1:5,000) and tubulin antibodies (10D8, 1:2000), obtained from Santa Cruz Biotech; and anti-K63 ubiquitin (HWA4C4, 1:750) and anti-Ubc13 antibodies (#371100, 1:1000), obtained from Enzo and Invitrogen, respectively. TRAF6 antibody (H-274) was also used for immunoprecipitation (1 g/sample). For ChIPs, the following antibodies were used at 2 g/sample: STAT3 (C-20) and c-Ets-1 antibodies (C-20), obtained from Santa Cruz Biotech; H3K4me3 (CS200580) and total H3 antibodies (04-928), obtained from Millipore; and Set1 antibody (A300-289A) from Bethyl Laboratories. For flow cytometry, fluorescently labeled antibodies to CD11b (M1/70, FITC-labeled), F4/80 (BM8, PerCP Cy5.5-labeled), M-CSFR (AFS98, APC-labeled) and RANK (R12-31, PE-labeled) from eBioscience were used at a 1:100 dilution. Recombinant murine RANKL, IL-6, IL-10 and IL-6 blocking antibody were purchased from R&D Systems.
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