The protein extracts were subjected to SDS-PAGE and were analyzed using the following primary antibodies: IL-6R; p-STAT3; STAT3 and β-actin (Abcam, Cambridge, MA, USA). The secondary antibodies were labeled with IRDye 700 (Rockland Immunochemicals, USA). The protein levels were detected by the Odyssey system (Li-Cor, Lincoln, NE, USA).
Il 6r
Manufactured by Abcam
Sourced in United States, Israel, United Kingdom
IL-6R is a protein that functions as a receptor for the cytokine interleukin-6 (IL-6). It plays a role in the regulation of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (2 mentions)
Irdye 700, by Rockland Immunochemicals (1 mentions)
Odyssey system, by LI COR (1 mentions)
Stat3, by Abcam (1 mentions)
P stat3, by Abcam (1 mentions)
Snail1, by Abcam (1 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
Cxcr4, by R&D Systems (1 mentions)
Pdgfb, by Santa Cruz Biotechnology (1 mentions)
Rad51, by Santa Cruz Biotechnology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
N cadherin, by Abcam (1 mentions)
Total stat3, by Cell Signaling Technology (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Pierce bca kit, by Thermo Fisher Scientific (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
6 protocols using il 6r
1
Profiling Immune Signaling Proteins
2
Comprehensive Immunofluorescence Antibody Panel
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Hoechst 33342 and anti-β-actin and anti-SDF-1 antibodies were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO). Draq5TM was purchased from eBioscience (San Diego, CA). Antibodies against ATR, Rad51, IL-6R, GP130, pATM (Ser1981), TWIST1, SNAIL1, N-cadherin and vimentin were from Abcam Inc. (Abcam, Cambridge, MA). Antibody against fibronectin was obtained from BD Pharmigen (BD Biosciences, San Diego, CA). ATM, AKT, pAKT (Ser 473), pATR (Ser428), ZEB1, SNAIL2 (SLUG), E-cadherin and β-catenin antibodies were purchased from Cell Signaling Technology Inc., (Danvers, MA). Antibodies against 53BP1 and γH2AX (Ser139) were obtained from EMD Millipore (Darmstadt, Germany). Rad51, PDGFB and ATR antibodies were from Santa Cruz biotechnology, (Dallas, TX). CXCR4, PDGFRα and PDGFRβ antibodies and human IL-6 ELIZA kit were from R&D (Minneapolis, MN). Secondary antibodies conjugated with Alexa® −488, −546, and −680 were from Molecular Probes (Invitrogen, Carlsbad, CA).
HSP90 inhibitor ganetespib [3–(2,4–dihydroxy–5–isopropylphenyl)–4–(1–methyl–1H–indol–5–yl)–1H– 1,2,4–triazol–5(4H)–one] was generously provided by Synta Pharmaceuticals Corp. (Lexington, MA).
HSP90 inhibitor ganetespib [3–(2,4–dihydroxy–5–isopropylphenyl)–4–(1–methyl–1H–indol–5–yl)–1H– 1,2,4–triazol–5(4H)–one] was generously provided by Synta Pharmaceuticals Corp. (Lexington, MA).
3
Protein Expression Analysis of Cell Signaling
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The protein lysates were harvested using RIPA lysis buffer, with quantification performed using the Pierce BCA kit (Thermo Fisher Scientific). For immunoblot analysis, 25-50 μg protein was boiled in Laemmli buffer and loaded on an 8-10% denaturing SDS-polyacrylamide gel and then transferred to PVDF membranes, which were blocked with 5% bovine serum albumin before immunoblotting with primary antibodies against IL-6R (#167742, Abcam), integrin β6 (Clone 442.5C4, Millipore Sigma), phospho-STAT-3 (#9145), total STAT-3 (#4904), phospho-ERK1/2 (#9106), phospho-AKT (#4060), Vimentin (#5741), E-cadherin (#3195) (Cell Signaling Technology), αv (#sc-9969), and β-actin (#sc-47778) (Santa Cruz). After the incubation with secondary antibodies, the bands were detected by enhanced chemiluminescence.
4
Quantitative Protein Analysis of Lung Tissues
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Total protein lysates were prepared from snap‐frozen lung tissues or cell lysates and subjected to ELISA and immunoblotting. Human and mouse IL‐6R ELISA sets were purchased from R&D Systems. Immunoblotting was performed with the following antibodies: total ADAM17 (from S. Rose‐John), pThr735‐ADAM17 (Sigma), Myc, pTyr705‐STAT3, pSer727‐STAT3, total STAT3, pSer473‐AKT, total AKT, pThr202/pTyr204‐ERK1/2, total ERK1/2, pThr180/pTyr182‐p38 MAPK, total p38 MAPK, pTyr1068‐EGFR, total EGFR, pSer82‐HSP27 (Cell Signaling Technology), cleaved Notch1 (Abcam), IL‐6R, Nrg1, pTyr1289‐ErbB3, total ErbB3, TGFα (Santa Cruz Biotechnology), and actin (Sigma). Protein bands were visualized using the Odyssey Infrared Imaging System (LI‐COR) and quantified using Image J. Antibody dilutions are indicated in Appendix Table S2 .
5
Western Blot Analysis of PD-L1 and IL-6R
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Protein extraction was performed using the protease inhibitor-contained RIPA buffer (R0010, Solarbio, Beijing, China). The protein sample was separated using freshly-prepared SDS-PAGE, electrotransferred onto PVDF membranes, and probed with primary antibodies: PD-L1 (ab228415, 1: 1000), IL-6R (ab128008, 1:1000), and β-actin (ab8226, 1:20000) (Abcam Inc.). Immunoblotting was visualized with goat anti-rabbit IgG (Transgene biology CO., Beijing, China), enhanced chemiluminescence detection reagents and captured under the Bio-Rad image system (Bio-Rad, USA). Gray value of the target protein bands was quantified using Quanity One software, with β-actin used for normalization.
6
In Situ Protein-Protein Interaction Assay
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The PLA method [35 (link)] was conducted to analyze the binding condition of cytokine to the cognate receptor in lung tissues, using a PLA kit (DuoLink Mouse Rabbit in situ PLA kit; Sigma-Aldrich). In brief, lung tissue sections were blocked and then incubated with the primary antibodies (i.e., TNF-α and TNFR1, IL-1β and IL-1R, or IL-6 and IL-6R; all from Abcam, Cambridge, UK), followed by incubation with the secondary antibody (i.e., the oligonucleotide-labeled PLA probe). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Images of all tissue sections were observed (DeltaVision Elite microscope; GE Healthcare, Marlborough, MA, USA) and scanned. The PLA signal intensities of the scanned images were measured using the image processing software ImageJ (a free software from https://imagej.nih.gov/ij/ , accessed on 11 July 2018).
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