Sb 505124

Manufactured by Merck Group

Sourced in United States, United Kingdom

SB-505124 is a selective inhibitor of the activin receptor-like kinase 5 (ALK5), which is a component of the transforming growth factor-beta (TGF-β) receptor complex. SB-505124 functions by blocking the TGF-β signaling pathway.

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Lab products found in correlation

Tgf β1, by R&D Systems (4 mentions) Necrostatin 1, by Merck Group (4 mentions) Z vad fmk, by R&D Systems (4 mentions) Ym155, by Selleck Chemicals (4 mentions) Sb431542, by Merck Group (3 mentions) Fetal bovine serum (fbs), by R&D Systems (3 mentions) Recombinant human tgf β1, by R&D Systems (3 mentions) Dulbecco modified eagle medium (dmem), by Merck Group (3 mentions) 5z 7 oxozeaenol, by Bio-Techne (2 mentions) Dexamethasone (dex), by Merck Group (2 mentions) β glycerophosphate, by Merck Group (2 mentions) Activin a, by R&D Systems (2 mentions) Tgf β1, by BioLegend (2 mentions) Crizotinib, by Merck Group (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Hepg2, by American Type Culture Collection (2 mentions) Sw620 cells, by American Type Culture Collection (2 mentions) Jsi 124, by Merck Group (2 mentions) Nsc33994, by Merck Group (2 mentions)

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SB-505124 compound (2 citations)

69 protocols using sb 505124

Nodal and Arp2/3 Signaling Disruption

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To block Nodal signalling, embryos were treated with SB505124 (S4696, Sigma-Aldrich, MI) soon after fertilization. In long-term treatment, embryos were allowed to develop in Millipore filtered seawater (MPFSW) at 50 µM SB505124 concentration until fixation at the mid-gastrula stage (6 hpf at 25°C), otherwise (short-term at 75 µM) triple washings were performed during the 32- to 64-cell stage (135–155 min postfertilization (mpf) at 25°C), and then embryos were also allowed to develop until fixation at the blastula (200 mpf), initial gastrula (4.5 hpf), mid-gastrula (6 hpf), late gastrula (7.5 hpf) and neurula (12–13 hpf) stages. For perturbing the active form of Arp2/3 complex, embryos were treated with CK666 (SML0006, Sigma-Aldrich) at 400 µM in MPFSW soon after fertilization and washed thoroughly three times at the two-cell stage (1 hpf at 25°C) in MPFSW. Embryos were allowed to develop until fixation at the blastula (200 mpf), initial gastrula (4.5 hpf), mid-gastrula (6 hpf) and late gastrula (7.5 hpf) stages. Treated embryos and those reared in MPFSW with the same volume of dimethyl sulfoxide (DMSO) added were fixed under the same protocol as for WISH specimens.

Morov A.R., Ukizintambara T., Sabirov R.M, & Yasui K. (2016). Acquisition of the dorsal structures in chordate amphioxus. Open Biology, 6(6), 160062.

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Acute Inflammatory Arthritis in Mice

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Acute inflammatory arthritis was induced in knee joints of C57BL/6N mice as previously described^{20 (link)} by intra-articular injection with 200 µg of mBSA (Sigma A-1009), followed by daily injections on days 0–2 with 250 ng of human IL-1β (BioLegend). At time of sacrifice, knee joints were isolated for histological analysis and qPCR. Draining popliteal and inguinal lymph nodes were isolated for flow cytometry analysis. The treatment with the TGF-βRI inhibitor SB-505124 (Sigma-Aldrich) was applied locally starting two hours before the induction of arthritis, and mice were injected intra-articularly on four consecutive days with either saline, 20% DMSO as vehicle control, or 75 nmol SB-505124 (Sigma-Aldrich) in 20% DMSO with an injection volume of 6 µl per joint. Anti-IL-17A antibodies (BioXCell) were used as positive control, administered as 50 µg antibody/mouse intra-peritoneally injected every other day (n = 8 mice/group). Mice were sacrificed by cervical dislocation four days after arthritis induction, and knee joints were subsequently isolated for histological analysis.

Aarts J., van Caam A., Chen X., Marijnissen R.J., Helsen M.M., Walgreen B., Vitters E.L., van de Loo F.A., van Lent P.L., van der Kraan P.M, & Koenders M.I. (2022). Local inhibition of TGF-β1 signaling improves Th17/Treg balance but not joint pathology during experimental arthritis. Scientific Reports, 12, 3182.

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RNA-seq and RT-PCR Protocol with SB505124

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For RNA-sequencing experiments, SB505124 from Sigma-Aldrich was used at a concentration of 100 μM in 12 mL embryo media in a 15 mm × 60 mm Petri dish (DaCosta Byfield et al., 2004 (link); Hagos et al., 2007 (link)). For RT-PCR and whole mount in situ hybridization, SB505124 from Tocris was used at a concentration of 20 μM, which caused a similar phenotype to 100 μM of the Sigma drug, in a total of 30 mL embryo media in a 100 mm × 20 mm Petri dish. For both SB505124 stocks, Dimethyl Sulfoxide (DMSO) was used to make the 10 mM stock solution of SB505124. SB505124 treatment/solution (12 mL for Sigma-Aldrich and 30 mL for Tocris, with a final concentration of 1% DMSO) was added to pooled groups of ZDR embryos at either sphere (4.0 hpf) or 30% epiboly (4.7 hpf) stages and left on until the embryos were snap frozen in liquid nitrogen or fixed in 4% paraformaldehyde. Control embryos were treated with 1% DMSO in embryo media and raised in parallel to inhibitor treated embryos. In addition, at least 20 embryos from each treatment group were left in SB505124 until 24 hpf and scored for pineal phenotype by whole mount in situ hybridization (WISH) to ensure that the embryos had the expected neural tube phenotype. Phenotypes at 24 hpf between both SB50124 stocks were similar at the concentrations used.

Kindt L.M., Coughlin A.R., Perosino T.R., Ersfeld H., Hampton M, & Liang J.O. (2018). Identification of Transcripts Potentially Involved in Neural Tube Closure Using RNA-sequencing. Genesis (New York, N.Y. : 2000), 56(3), e23096.

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Urate-Primed Monocyte Cytokine Secretion

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For urate priming experiments, adherent monocytes were primed for 24 h in RPMI supplemented with 10% human pool serum with or without urate (Sigma, 69-93-2) and recombinant TGF-β1 (R&D Systems, Catalogue number 7754-BH-005). After 24 h, cells were restimulated with 10 ng/mL ultra-pure E. coli LPS (InVivogen, Catalogue number tlrl-pelps). Subsequently, cell-free supernatants were collected. Secretion of cytokines was measured in supernatants using ELISA kits for IL-1β, IL-6, IL-1Ra and TGF-β (R&D Systems, Catalogue number DY201, DY206, DY280 and DY240 respectively).
To inhibit TGF-β receptor signalling, three inhibitors were used. The ALK4/5/7-kinase inhibitor SB-505124 (Sigma) in a concentration of 5 μM with DMSO as solvent control, 5Z-7-oxozeaenol (100 nM) dissolved in DMSO (Tocris Bioscience) and a blocking antibody against TGF-β receptor II (AF-241-NA, R&D systems) with mouse IgG1 as the isotype control (10 μg/mL). Cells were pre-incubated with the inhibitor for 0.5 h before adding urate.

Klück V., Cabău G., Mies L., Bukkems F., van Emst L., Bakker R., van Caam A., Crişan T.O, & Joosten L.A. (2023). TGF-β is elevated in hyperuricemic individuals and mediates urate-induced hyperinflammatory phenotype in human mononuclear cells. Arthritis Research & Therapy, 25, 30.

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Progesterone and Inflammation Regulation

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Progesterone (4 pregnene-3,20-dione, P4), Lipopolysaccharide (LPS, from Escherichia coli 055:B5), Mifepristone (RU-486) and SB-505124 (S4696) were purchased from Sigma (St Louis, MO, USA).

Canciello A., Russo V., Berardinelli P., Bernabò N., Muttini A., Mattioli M, & Barboni B. (2017). Progesterone prevents epithelial-mesenchymal transition of ovine amniotic epithelial cells and enhances their immunomodulatory properties. Scientific Reports, 7, 3761.

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Osteogenic Differentiation of SV-HFO Cells

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Human SV‐HFO cells (Simian virus 40‐immortalized osteoblast precursors) were cultured (1 × 10⁴ vital cells/cm²) in α‐MEM (pH 7.5, phenol‐red free; GIBCO, Paisley, UK) supplemented with streptomycin/penicillin, 1.8 mM CaCl₂ (Sigma, St. Louis, MO), HEPES, and 2% charcoal‐treated and heat inactivated fetal bovine serum (GIBCO). After 2 days, medium was supplemented with dexamethasone (100 nM) and β‐glycerophosphate (10 mM; Sigma, St. Louis, MO) to induce osteogenic differentiation. Activin‐A (50 ng/ml; R&D System, Minneapolis, MN) was added at Day 5 of osteogenic differentiation and removed after 2 days, keeping cultures in osteogenic medium. Medium was replaced every 2–3 days.
To assess the specificity of the Activin‐A signaling, osteoblasts were treated with a SMAD inhibitor (SB‐505124; Sigma‐Aldrich, St. Louis, MO). SB‐505124 selectively inhibits ALK4 kinase activity, repressing Activin A‐ and TGF‐β‐induced SMAD2 and SMAD3 signaling (Harrison et al., 2005). SB‐505124 (0.125 µM in dimethyl sulfoxide) was added 30 min before Activin‐A, and removed at the same time. Osteoblasts treated only with SB‐505124 were used as control.

Baroncelli M., Drabek K., Eijken M., van der Eerden B.C., van de Peppel J, & van Leeuwen J.P. (2019). Two‐day‐treatment of Activin‐A leads to transient change in SV‐HFO osteoblast gene expression and reduction in matrix mineralization. Journal of Cellular Physiology, 235(5), 4865-4877.

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Phosphorylated Smad2/3 Expression in Murine Synovial Tissue

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Protein expression of phosphorylated Smad2/3 was evaluated on 7 µm paraffin-embedded sections of murine synovial tissue (n = 3 per group), collected 1 h after (co-)injection of recombinant 10 ng TGF-β1 (Biolegend) with or without 75 nmol SB-505124 (Sigma-Aldrich) into the knee joints of naïve C57BL/6 N mice. For immunohistochemistry, endogenous peroxidase activity was blocked with 3% H₂O₂ (Merck Millipore) in methanol, and antigen retrieval was performed in 10 mM citrate buffer, pH 6.0 at 60 °C. Subsequently, sections were stained with primary antibodies: rabbit anti-mouse pSmad2/3 (Cell Signaling #3108) (1:300 for 60 min at RT), mouse or isotype. Subsequently, the sections were stained with biotinylated anti-mouse IgG H + L (1:100 for 30 min at RT). Next, a biotin-streptavidin detection system was used according to the manufacturer’s protocol (PK-6101; Vector Laboratories). Peroxidase was developed with diaminobenzidine (Sigma-Aldrich) and counterstained with hematoxylin for 60 s.

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Selective TGF-β receptor 1 kinase inhibition

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TGF-β receptor 1 kinase was selectively blocked using the kinase inhibitor SB-505124 (Sigma-Aldrich, Buchs, Switzerland). The compound was reconstituted in DMSO to obtain stock solutions of 0, 0.3, 0.75, 1.5, or 3 mM, and added to the monolayer and pellet cultures at concentrations of 0, 100, 250, 500, or 1000 nM, respectively. At each medium change, the culture medium was supplemented with the inhibitor.

Tekari A., Luginbuehl R., Hofstetter W, & Egli R.J. (2015). Transforming Growth Factor Beta Signaling Is Essential for the Autonomous Formation of Cartilage-Like Tissue by Expanded Chondrocytes. PLoS ONE, 10(3), e0120857.

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Inhibition of TGF-β Signaling Pathways

Cited 1 time

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Before stimulation, cells were deprived of serum for 24 h and thereafter stimulated with 1 ng/ml recombinant human TGFβ1 (Biolegend, the Netherlands) for 2 or 24 h. In experiments where inhibitors were used, DMSO was used as vehicle control. To block TAK1 activity, we used (5Z)-7-Oxozeaenol [17 (link)] (Tocris Bioscience) in a concentration of 0.5 μM. To inhibit ALK5 kinase, we used SB-505124 [15 (link)] (Sigma Aldrich) in a concentration of 5 μM. For inhibition of ALK1 kinase, LDN-193189 [14 (link)] (Axon Medchem) was used in a concentration of 0.05 μM. This concentration of LDN-193189 is well above its reported half maximal inhibitory concentration (IC₅₀), 0.8 nM for ALK1, but far below its IC₅₀ for ALK5 of 350 nM [16 (link), 23 (link)]. Cells were pre-incubated with the inhibitors for 1 h prior to addition of TGF-β1. Either 2 or 24 h after addition TGF-β1, medium was removed and TRI-reagent was added for RNA isolation.

van Caam A., Madej W., Garcia de Vinuesa A., Goumans M.J., ten Dijke P., Blaney Davidson E, & van der Kraan P. (2017). TGFβ1-induced SMAD2/3 and SMAD1/5 phosphorylation are both ALK5-kinase-dependent in primary chondrocytes and mediated by TAK1 kinase activity. Arthritis Research & Therapy, 19, 112.

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Gastric and Colorectal Cancer Cell Lines

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Human gastric cancer MKN45 cells and MKN28 cells (RIKEN, Japan), AGS cells (ATCC, Manassas, VA), SGC-7901 cells and mouse gastric cancer MFC cells (Cell Bank, Shanghai), human colon cancer SW620 cells and human liver cancer cells HepG2 (ATCC, Manassas, VA), were maintained in RPMI-1640 media containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco BRL). BMFs (EGFP⁺) that were freshly isolated from gastric dysplastic tissues of IL-1β transgenic mice ^{29 (link)} transplanted with EGFP⁺ bone marrow according to our previous method ^{25 (link)}. All cell lines were tested for mycoplasma by a PCR method (Stratagene), and all cell lines were mycoplasma negative. BMFs within 12 generations were used. JSI-124 and NSC33994 (JAK2 inhibitor), crizotinib (c-Met inhibitor) and SB-505124 (TGF-β1 type I receptor inhibitor) were purchased from Sigma and Tocris (R&D Systems), dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. Human (h) and mouse (m) recombinant (r) IL-6 and HGF purchased from Peprotech (Rocky Hill,NJ). The anti-mouse IL-6 neutralizing antibody (Cat. MAB406) and anti-human TGF-β1 antibody (Cat. MAB7364) neutralizing antibodies were purchased from R&D Systems (Minneapolis, MN, Indiana).

Zhu L., Cheng X., Shi J., Jiacheng L., Chen G., Jin H., Liu A.B., Pyo H., Ye J., Zhu Y., Wang H., Chen H., Fang J., Cai L., Wang T.C., Yang C.S, & Tu S.P. (2016). Crosstalk between bone marrow-derived myofibroblasts and gastric cancer cells regulates cancer stemness and promotes tumorigenesis. Oncogene, 35(41), 5388-5399.

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