Anti gp130

Manufactured by Cell Signaling Technology

Sourced in Germany

Anti-GP130 is a laboratory reagent used in cell biology research. It is an antibody that binds to the GP130 protein, which is a component of the receptor complex for multiple cytokines, including IL-6, IL-11, and LIF. The antibody can be used to detect and study the expression and function of GP130 in cells and tissues.

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4 protocols using anti gp130

Plasma Protein Profiling using Western Blot

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Mouse plasma samples were diluted 1:1000 in phosphate-buffered saline (PBS). Control BL/6 mouse plasma was either untreated (intact glycosylation) or deglycosylated with N-Glycanase (PROzyme) per the manufacturer’s protocol. Cell lines were grown as indicated and lysed in equal volumes of RIPA lysis buffer (Boston Bioproducts) containing protease and phosphatase inhibitors (Roche). Briefly, 20 μl of each diluted plasma sample or equal amounts of cell culture extract was boiled for 10 min in 1× loading buffer (Boston Bioproducts). Samples were electrophoresed on a 4–15% gradient gel (Bio-Rad), transferred to nitrocellulose membranes (Invitrogen) and probed for antigens of interest. The following primary antibodies were used: anti-Transferrin (Abcam, ab82411); biotinylated anti-ALS (R&D Systems, BAF1436); anti-ICAM-1 (Santa Cruz Biotechnology, sc-8439); anti-GP130 (Cell Signaling Technology, CST-3732); anti-PMM2 (Abcam, ab138817); anti-β-actin (Cell Signaling Technology, CST-3700) and anti-β-tubulin (Cell Signaling Technology, CST-2128). Antigens were visualized using IRDye secondary antibodies and an Odyssey Imager (both LI-COR Biosciences).

Chan B., Clasquin M., Smolen G.A., Histen G., Powe J., Chen Y., Lin Z., Lu C., Liu Y., Cang Y., Yan Z., Xia Y., Thompson R., Singleton C., Dorsch M., Silverman L., Su S.S., Freeze H.H, & Jin S. (2016). A mouse model of a human congenital disorder of glycosylation caused by loss of PMM2. Human Molecular Genetics, 25(11), 2182-2193.

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Pancreatic Cancer Cell Line Characterization

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The human PDAC cell lines L3.6pl and AsPC-1 were used for in vitro and in vivo studies. AsPC-1 cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). L3.6pl is a secondary highly metastatic human pancreatic adenocarcinoma cell line derived from an orthotopic mouse xenograft model [15 ]. All cell lines were maintained in Dulbecco’s Minimal Essential Medium (Invitrogen GmbH, Karlsruhe, Germany), supplemented with 10% fetal bovine serum and 1% penicillin streptomycin and were incubated in a humidified atmosphere of 5% CO₂ at 37 °C.
Raloxifene was purchased from Sigma-Aldrich (Schnelldorf, Germany) and was dissolved in 0.1% dimethyl sulfoxide (DMSO, Carl Roth GmbH & Co. KG, Karlsruhe, Germany). IL-6 was purchased from Invitrogen GmbH (Karlsruhe, Germany) and was dissolved in acetic acid. Anti-STAT3, anti-phosphorylated-STAT3 (Y705) and anti-gp130 antibodies were obtained from Cell Signaling Technology (Frankfurt am Main, Germany).

Pozios I., Seel N.N., Hering N.A., Hartmann L., Liu V., Camaj P., Müller M.H., Lee L.D., Bruns C.J., Kreis M.E, & Seeliger H. (2020). Raloxifene inhibits pancreatic adenocarcinoma growth by interfering with ERβ and IL-6/gp130/STAT3 signaling. Cellular Oncology (Dordrecht), 44(1), 167-177.

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Immunoblotting Analysis of Signaling Pathways

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The cells were lysed with NP-40 lysis buffer and centrifuged at 15,000 rpm for 15 min at 4 °C. The supernatants were collected, and total protein content was measured using the Bradford assay (Bio-Rad Laboratories). Lysates were separated with Bolt 4 to 12 % Bis-Tris polyacrylamide gels (Thermo Fisher Scientific), transferred to membrane filters, and subjected to immunoblotting using anti-GP130 (1:2000, Cell Signaling, #3732), anti-LIFR (1:1000, Santa Cruz Biotechnology, A-10), anti-EGFR (1:1000, Cell Signaling, #2232), anti-p-EGFR-Tyr1068 (1:1000, Cell Signaling, #2234), anti-AKT (1:1000, Cell Signaling, #4691), anti-p-AKT-Ser473 (1:1000, Cell Signaling, #4060), anti-ERK1/2 (1:1000, Cell Signaling, #9102), anti-p-ERK1/2-Thr202/Tyr204 (1:1000, Cell Signaling, #9101), anti-MEK1/2 (1:1000, Cell Signaling, #8727), anti-p-MEK1/2-Ser217/221 (1:1000, Cell Signaling, #9154), anti-Stat3 (1:1000, Cell Signaling, #4904), anti-p-Stat3-Tyr705 (1:1000, Cell Signaling, #9145), anti-Jak1 (1:1000, Cell Signaling, #3344), anti-p-Jak1-Tyr1034/1035 (1:1000, Cell Signaling, #74129), anti-Jak2 (1:1000, Cell Signaling, #3230), anti-p-Jak2-Tyr1008 (1:1000, Cell Signaling, #8082), anti-Jak3 (1:1000, Cell Signaling, #8827), anti-p-Jak3-Tyr980/981 (1:1000, Cell Signaling, #5031), and anti-beta-tubulin mouse antibody (1:5000, Sigma).

Hara T., Chanoch-Myers R., Mathewson N.D., Myskiw C., Atta L., Bussema L., Eichhorn S.W., Greenwald A.C., Kinker G.S., Rodman C., Castro L.N., Wakimoto H., Rozenblatt-Rosen O., Zhuang X., Fan J., Hunter T., Verma I.M., Wucherpfennig K.W., Regev A., Suvà M.L, & Tirosh I. (2021). Interactions between cancer cells and immune cells drive transitions to mesenchymal-like states in glioblastoma. Cancer cell, 39(6), 779-792.e11.

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Cell Culture and Reagent Preparation for PDAC Research

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AsPC-1 cells were purchased from the American Type Cell Culture Collection (Virginia, USA). L3.6pl is a secondary, highly metastatic human PDAC cell line of an orthotopic mouse xenograft model (Bruns et al. 1999 (link)). Cell lines were maintained in Dulbecco’s Minimal Essential Medium (Invitrogen GmbH, Karlsruhe, Germany), supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. All cells were incubated in a humidified atmosphere of 5% CO₂ at 37 °C. Cells were routinely checked for mycoplasma contamination using PlasmoTest (InvivoGen).
SC144 (a quinoxalinhydrazide derivative) was purchased from Sigma-Aldrich (Schnelldorf, Germany) and was solved in 0.1% dimethyl sulfoxide (DMSO). IL-6 was purchased from Invitrogen GmbH (Karlsruhe, Germany) and was dissolved in acetic acid. OSM was obtained from Cell Signaling Technology (Frankfurt am Main, Germany), and dissolved in phosphate-buffered saline. Anti-phosphorylated-STAT3 (Y705), anti-STAT3, and anti-gp130 antibodies were purchased from Cell Signaling Technology (Frankfurt am Main, Germany). Anti-β-actin was purchased from Sigma–Aldrich (Taufkirchen, Germany).

Pozios I., Hering N.A., Guenzler E., Arndt M., Elezkurtaj S., Knösel T., Bruns C.J., Margonis G.A., Beyer K, & Seeliger H. (2022). Gp130 is expressed in pancreatic cancer and can be targeted by the small inhibitor molecule SC144. Journal of Cancer Research and Clinical Oncology, 149(1), 271-280.

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