Sb431542

Manufactured by Cell Signaling Technology

Sourced in United States

SB431542 is a potent and selective inhibitor of the transforming growth factor-beta (TGF-β) type I receptor kinases ALK4, ALK5, and ALK7. It inhibits the phosphorylation and activation of Smad2 and Smad3, which are key intracellular mediators of the TGF-β signaling pathway.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Sp600125, by Cell Signaling Technology (2 mentions) Sb203580, by Cell Signaling Technology (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Collagen 3, by Abcam (1 mentions) Gapdh, by Abcam (1 mentions) Uo126, by Cell Signaling Technology (1 mentions) Human recombinant tgf β1, by Santa Cruz Biotechnology (1 mentions) Antibodies against α sma, by Abcam (1 mentions) Phospho c jun, by Cell Signaling Technology (1 mentions) Mk 2206, by Selleck Chemicals (1 mentions) Ve cadherin, by Abcam (1 mentions) Recombinant human tgf β1, by Thermo Fisher Scientific (1 mentions) Sp600125, by Merck Group (1 mentions) Sb203580, by Merck Group (1 mentions) Bay 11 7085, by Cayman Chemical (1 mentions) Gw441756, by Selleck Chemicals (1 mentions) Tnf α, by Miltenyi Biotec (1 mentions) Inverted microscope, by Olympus (1 mentions) Bronchial epithelial growth medium (begm), by Lonza (1 mentions)

9 protocols using sb431542

TGF-β1 Signaling Pathway Investigation

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Human recombinant TGF‐β1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against α‐SMA, VE‐cadherin, collagen I, collagen III, CD31, vWF and GAPDH were purchased from Abcam (Cambridge, MA, USA). Antibodies against phospho‐Smad 2 (Ser465/467), phosphor‐Smad 3 (Ser423/425), phospho‐Akt (Ser473), phospho‐mTOR (Ser2448), phospho‐p70S6K (Thr389), phospho‐Erk 1/2 (Thr202/Tyr204), phospho‐p38 MAPK (Thr180/Tyr182), phospho‐c‐Jun (Ser73), Smad 2, Smad 3, Akt, mTOR, p70S6K, Erk 1/2, p38 MAPK and c‐Jun, and selective inhibitors including SB431542, SB203580, UO126 and SP600125 were purchased from Cell Signaling Technology (Beverly, MA, USA). MK2206 was purchased from Selleckchem (Houston, TX, USA). Penicillin‐streptomycin and Roswell Park Memorial Institute (RPMI)‐1640 medium were purchased from Invitrogen (Life Technologies, Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA, USA).

Wang Z., Han Z., Tao J., Wang J., Liu X., Zhou W., Xu Z., Zhao C., Wang Z., Tan R, & Gu M. (2017). Role of endothelial‐to‐mesenchymal transition induced by TGF‐β1 in transplant kidney interstitial fibrosis. Journal of Cellular and Molecular Medicine, 21(10), 2359-2369.

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Signaling Pathway Inhibitors in Cell Culture

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Recombinant human TGF-β1 was obtained from PeproTech, Inc. (Rocky Hill, NJ, USA), recombinant rat NGF was obtained from Alomone Labs (Jerusalem, Israel), and recombinant rat IL-1β and TNF-α were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). SB-431542, a TGF-β type I receptor inhibitor, was supplied by Cell Signaling Technology, Inc. (Beverly, MA, USA). The p38 MAPK inhibitor SB203580, JNK inhibitor SP600125, and Smad3 inhibitor SIS3 (33 (link)) were obtained from Merck KGaA (Calbiochem; Darmstadt, Germany). Adenosine 5′-O-(3-thio) triphosphate (ATPγS; ab138911) was purchased from Abcam (Cambridge, UK). The TrkA inhibitor GW441756 was purchased from Selleck Chemicals (Houston, TX, USA), while the NF-κB inhibitor BAY 11-7085 was from Cayman Chemical (Ann Arbor, MI, USA).

Ohta M., Chosa N., Kyakumoto S., Yokota S., Okubo N., Nemoto A., Kamo M., Joh S., Satoh K, & Ishisaki A. (2018). IL-1β and TNF-α suppress TGF-β-promoted NGF expression in periodontal ligament-derived fibroblasts through inactivation of TGF-β-induced Smad2/3- and p38 MAPK-mediated signals. International Journal of Molecular Medicine, 42(3), 1484-1494.

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Inducing EMT in BEAS-2B Cells

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BEAS-2B cells derived from the normal human bronchial epithelium were purchased from ATCC and cultured in bronchial epithelial cell growth medium (BEGM, Lonza) at 37 °C in a humidified 5% CO₂ atmosphere. Culture flasks should be precoated with a mixture of 0.01 mg/mL fibronectin, 0.03 mg/mL collagen, and 0.001 mg/mL bovine serum albumin dissolved in the medium according to the ATCC recommended protocol. To induce EMT, BEAS-2B cells were seeded at 80% confluence a day before the stimulation of P. aeruginosa’s LPS (Sigma). BEAS-2B cells were pretreated with Integrin αvβ6–blocking antibody (10D5, Abcam) or TGF-β1-Smad2/3 signaling inhibitor, SB431542 (Cell Signaling), for 2 h prior to incubation with 2 μg/mL P. aeruginosa’s LPS (Gong et al. 2014 (link)). Morphologic images were captured with an Olympus Inverted Microscope.

Liu W., Sun T, & Wang Y. (2019). Integrin αvβ6 mediates epithelial-mesenchymal transition in human bronchial epithelial cells induced by lipopolysaccharides of Pseudomonas aeruginosa via TGF-β1-Smad2/3 signaling pathway. Folia Microbiologica, 65(2), 329-338.

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Chemokinetic Migration of Fibroblasts

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Chemokinetic migration of fibroblasts was assayed by using modified Boyden chamber and cell scratch assays. Boyden chamber assay was performed as described by Bond et al [22 (link)] using 3T3 treated with 0.01μM AngII or 5ng/ml TGFβ and cell scratch was performed using Human Scar Fibroblasts (HSF) treated in 1μM AngII or 20ng/mL TGFβ as these substrate concentrations were found to elicit the most significant response for the respective cell types and experimental conditions. To conduct the cell scratch assay, HSF were plated onto 24-well chambers, 3 chambers per treatment group. At 80–90% confluence, a scratch was made with a plastic P20 pipet tip, chambers were rinsed, and incubated with inhibitors (ALK5-20μΜ SB431542, MEK-10μM U0126 (Cell Signaling, Danvers, MA), p38-10μM SB203580 (Cell Signaling), JNK-10μM SP600125), 10μM Losartan, or DMSO control in DMEM for 1h prior to TGFβ or AngII treatment. Images were taken of scratch area at 0h, 24h, and 48h using 4× air objective, measured using ImageJ freehand measure tool, and area of scratch closure calculated as a fraction of initial size.

Ehanire T., Ren L., Bond J., Medina M., Li G., Bashirov L., Chen L., Kokosis G., Ibrahim M., Selim A., Blobe G.C, & Levinson H. (2014). Angiotensin II Stimulates Canonical TGFβ Signaling Pathway through Angiotensin Type 1 Receptor to Induce Granulation Tissue Contraction. Journal of molecular medicine (Berlin, Germany), 93(3), 289-302.

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Mechanical Compression and TGF-β Signaling in HBE Cells

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Poly(I:C) (Invivogen, San Diego, CA) or recombinant human (rh) TGF-β1 (10 ng/ml, Cell Signaling Technologies, Danvers, MA) was spiked into the basolateral media of HBE cells in ALI cultures [26 (link), 27 (link)]. PBS for Poly(I:C) or sodium citrate for rhTGF-β1 was used as vehicle control. As previously described [23 (link), 24 (link), 32 (link)], HBE cells were subjected to mechanical compression at a magnitude of 30 cm H₂O pressure for three hours. Time-matched controls were subjected to 0 cm H₂O pressure. In experiments where a pharmacological inhibitor of TGF-β receptor 1, SB431542 was used (10 μM, Cell Signaling Technology), this was spiked to the basolateral medium at a final concentration of 10 μM, one hour prior to exposure to either stimulation. As a vehicle control for SB431542, 0.1% DMSO was used.

Dy A.B., Girkin J., Marrocco A., Collison A., Mwase C., O’Sullivan M.J., Phung T.K., Mattes J., Koziol-White C., Gern J.E., Bochkov Y.A., Bartlett N.W, & Park J.A. (2023). Rhinovirus infection induces secretion of endothelin-1 from airway epithelial cells in both in vitro and in vivo models. Respiratory Research, 24, 205.

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HUVEC Culture and DHA/SB431542 Treatment

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HUVECs were obtained from American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc.) and antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin). The cells were cultured in humidified air at 37˚C with 5% CO₂. DHA was obtained from Sigma-Aldrich (Merck KGaA), and SB431542 was purchased from Cell Signaling Technology, Inc. They were both dissolved at 10 mM in dimethyl sulfoxide.

Guo L., Wen X., Hou Y., Sun R., Zhang L., Liu F, & Liu J. (2021). Dihydroartemisinin inhibits endothelial cell migration via the TGF-β1/ALK5/SMAD2 signaling pathway. Experimental and Therapeutic Medicine, 22(1), 709.

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Activin/Smad2/3 Signaling in Sensory Neurons

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For gain of function experiment, either 10ng/ml of recombinant Activin A, B and AB (R&D, USA) or PBS containing 0.1% BSA was added into myelin coated eight chamber slides with 3,000 naïve or pre-conditioned C57BL/6 sensory neurons. For loss of function experiment, 3,000 naïve and pre-conditioned CAST/Ei sensory neurons were cultured on a myelin for 24 hours with 1% ethanol vehicle, 100nM SB 431542 (TOCRIS, USA).
Nuclear Smad2/3 counts: C57BL/6 and CAST/Ei DRG naïve and preconditioned DRG neurons cultured with or without the presence of either Activin or SB-431542 were assayed for nuclear pSmad2/3 expression by immunostaining (Cell Signaling, #8828). Cells were defined as either containing no, low or strong pSmad2/3 signal within the nucleus. Percentage of the cells lacking nuclear pSmad2/3 were compared. The values given are the averages of at least three independent experiments counted blind to treatment.

Omura T., Omura K., Tedeschi A., Riva P., Painter M.W., Rojas L., Martin J., Lisi V., Huebner E.A., Latremoliere A., Yin Y., Barrett L., Singh B., Lee S., Crisman T., Gao F., Li S., Kapur K., Geschwind D.H., Kosik K.S., Coppola G., He Z., Carmichael S.T., Benowitz L.I., Costigan M, & Woolf C.J. (2015). Robust axonal regeneration occurs in the injured CAST/Ei mouse central nervous system. Neuron, 86(5), 1215-1227.

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Silencing Vash1 in Rat Renal Mesangial Cells

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In order to ensure the effectiveness of transfection, we used small interfering RNA (siRNA) for silencing of rat Vash1 mRNA (GenBank no. NM_503052) designed and synthesized by GenePharma (Shanghai, China). According to the instructions of the transfection reagent, three different sequences (siRNA-V1, V2, and V3, Table 1) and non-specific siRNA (siRNA-V) were used as controls. Cells were plated into six-well plates and after 24 h starvation synchronization with Opti-MEM, they were transfected with 100 pmol/4 μg siRNA. Meanwhile, RMCs were also transiently transfected with Sirt1 siRNA or Hif1a siRNA (sc-108043 or sc-45919, Santa Cruz, CA) by Lipofectamine 2000 (Invitrogen, USA), based on our previous research (Huang et al., 2014 (link)). Six hours after transfection, cells were treated with normal or high glucose media. Moreover, cells were cultured with a signal inhibitor to suppress rat TGFβ1/Smad3 pathway. The inhibitor is called SB431542 (10 μmol/L, #14775) and was designed as well as synthesized by Cell Signaling Technology as described in previous studies (Kanwar et al., 2011 (link); Kanomata et al., 2013 (link)).

Ren H., Shao Y., Wu C., Lv C., Zhou Y, & Wang Q. (2020). VASH-1 Regulates Oxidative Stress and Fibrosis in Diabetic Kidney Disease via SIRT1/HIF1α and TGFβ1/Smad3 Signaling Pathways. Frontiers in Molecular Biosciences, 7, 137.

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Western Blot Protein Analysis

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Reagents were purchased from Sigma-Aldrich (MO, USA) unless otherwise specified. The antibodies used in western blotting are described in each section. Doxorubicin hydrochloride was purchased from Sigma-Aldrich (MO, USA). LY294002 (PI3K inhibitor) and SB431542 (TGF-β/Smad inhibitor) were purchased from Cell Signaling (MA, USA).

Narikawa M., Umemura M., Tanaka R., Hikichi M., Nagasako A., Fujita T., Yokoyama U., Ishigami T., Kimura K., Tamura K, & Ishikawa Y. (2019). Doxorubicin induces trans-differentiation and MMP1 expression in cardiac fibroblasts via cell death-independent pathways. PLoS ONE, 14(9), e0221940.

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