Sb431542

Manufactured by Selleck Chemicals

Sourced in United States, China, France, Germany

SB431542 is a small molecule inhibitor that selectively targets the transforming growth factor-beta (TGF-β) superfamily type I activin receptor-like kinases (ALK4, ALK5, and ALK7). It blocks the phosphorylation of SMAD2 and SMAD3, which are key mediators of the TGF-β signaling pathway.

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Lab products found in correlation

202 protocols using sb431542

Lineage-Specific Differentiation of hESCs

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Lineage-specific differentiation of hESCs was conducted as previously described [19 (link)]. Briefly, hESCs were passaged to 12-well plates at a ratio of 1:12. Differentiation starts on day 2 after passaging for endoderm differentiation and starts on day 1 after passaging for all the other lineages. For trophoblast differentiation, cells were differentiated in E7 medium (E8 medium without FGF2) with 20 ng/ml BMP4 for 6 days with medium change every 2 days and then harvested on day 6 for RT-qPCR analysis of CGA, CGB, GCM1, GATA2 and TROP2. For mesoderm differentiation, cells were differentiated in E8 medium supplemented with 20 ng/ml BMP4 for 2 days and harvested on D2 for RT-qPCR analysis of TBXT and MIXL1 expression. For endoderm differentiation, cells were treated with 5 uM CHIR in E8 for 24 h and then changed to E8 medium supplemented with activin A (10 ng/ml) for 3 days. Expression of SOX17 and FOXA2 was analyzed by RT-qPCR. For ectoderm differentiation, cells were treated with E6 medium (E8 without FGF2 and TGFβ) with 10 μM SB431542 (Selleck) for 1 day and subsequently treated with 10 μM SB431542 and 100 nM LDN193189 (Selleck) in E6 medium for 4 days. Expression levels of PAX3 and PAX6 were examined by RT-qPCR.

Deng C., Zhang Z., Xu F., Xu J., Ren Z., Godoy-Parejo C., Xiao X., Liu W., Zhou Z, & Chen G. (2022). Thyroid hormone enhances stem cell maintenance and promotes lineage-specific differentiation in human embryonic stem cells. Stem Cell Research & Therapy, 13, 120.

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Isolating and Modulating Cardiomyocytes

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Adult ventricular cardiomyocytes (ACMs) were isolated from adult mice described previously.²¹ (link) Neonatal mouse/rat ventricular CMs were isolated from 1-day-old C57BL/6J mice or Sprague-Dawley rats. Neonatal mouse cardiomyocyte (NMCM) hypertrophy was induced with phenylephrine (PE) (50 μM) for 24 hours, and cell size was determined by measuring cell surface area, hypertrophic, and fibrotic markers. CMs were cultured in serum-free medium for 6 hours and then stimulated separately with 50 ng/mL rGDF-11 for 24 hours, 1 μM inhibitor of activin receptor-like kinase (ALK) SB431542 for 24 hours, and 1 μM SB431542 for 24 hours after GDF-11 stimulation or 10 μM Akt inhibitor MK 2206 for 24 hours (Selleckchem).

Zhu J., Zhang N., Zhao Y., Liu Q., Wang Y., Chen M., Ma Q., Dong A., Wang Y, & Yu H. (2023). Deficiency of GDF-11 Accelerates TAC-Induced Heart Failure by Impairing Cardiac Angiogenesis. JACC: Basic to Translational Science, 8(6), 617-635.

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Transcriptional Inhibition Assay in Halocynthia Embryos

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Actinomycin D (50-76-0; A1410; Sigma) was diluted into DMSO at 10 mg/mL stock. The stock solution was diluted into ASW to a final concentration of 40 μg/mL. This concentration was reported to block transcription in Halocynthia embryos (Miyaoku et al, 2018 (link)). Flavopiridol (146426-40-6; S1230; Selleck chemicals) was diluted into water to 10 mM stock. The stock solution was diluted into ASW to final concentration 1 and 10 μM. The transcriptional inhibitor-treated embryos were fixed by MEM-PFA (4% PFA, 0.1 M MOPS, 0.5 M NaCl, 1 mM EGTA, 2 mM MgSO₄) after 1 h inhibitor treatment, and used for in situ hybridization.
1-Azakenpaullone (S7193; Selleckchem; Feinberg et al, 2019 (link)), Ruxolitinib (INCB018424; S1378; Selleckchem), Vismodegib (GDC-0449; S1082; Selleckchem), DAPT (208255-80-5; D5942; Millipore Sigma), SB431542 (S1067; Selleckchem; Ohta and Satou, 2013 (link)), U0126 (9903; Cell Signaling Technology; (Hudson et al, 2003 (link))) and Dorsomorphin (1219168-18-9; S7306; Selleckchem; (Ohta and Satou, 2013 (link); Feinberg et al, 2019 (link))) were used to perturb define signaling pathways as described in corresponding references. These treatments were done in a final concentration of 10 μM for 2 or 4 h. The inhibitor-treated embryos were fixed by MEM-PFA after 2 h inhibitor treatment, and used for in situ hybridization.

Ohta N, & Christiaen L. (2024). Cellular remodeling and JAK inhibition promote zygotic gene expression in the Ciona germline. EMBO Reports, 25(5), 2188-2201.

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Maintenance of Human iPSC-Derived NPCs

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Human iPSC/iPSC-derived NPCs were provided by IxCell Biotechnology Ltd. The human iPSCs-derived NPCs cells were maintained as an adherent culture in 50% DMEM-F12 and 50% Neurobasal Medium, containing N2 supplement, B27 supplement (Minus Vitamin A), NEAA, 1 × Glutamax, 10 ng/mL FGF-Basic Recombinant Human Protein (bFGF, Gibco, New York, NY, USA), 10 ng/mL LIF Recombinant Human Protein (hlif, Gibco, New York, NY, USA), 5 μM SB431542 (Selleckchem, Shanghai, China), 3 μM CHIR99021 (Selleckchem, Shanghai, China), and 200 μM L-Ascorbic acid 2-phosphatesesquimagnesium salt hydrate (Sigma, St. Louis, MO, USA). The cells were maintained at 37 °C in humidified air with 5% CO₂.

Zhang R., Lu J., Pei G, & Huang S. (2023). Galangin Rescues Alzheimer’s Amyloid-β Induced Mitophagy and Brain Organoid Growth Impairment. International Journal of Molecular Sciences, 24(4), 3398.

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Prostate Cancer Cell Line Maintenance

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Human PC3 and DU145 cells were obtained from ATCC (Manassas, VA). Cells were maintained in DMEM high glucose medium (Hyclone, Logan, UT) with 10% FBS (Atlanta Biologicals, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified incubator at 37°C and 5% CO₂, and routinely passaged when 80–90% confluent. Antibodies for p β-catenin, β-catenin, E-cadherin, and N-cadherin were purchased from Cell Signaling (Danvers, MA). Anti-TGFβ-RII was purchased from Abcam (Cambridge, MA). Anti-β-actin was purchased from Sigma (St. Louis, MO). Compound inhibitors such as TCBN, ICG001, IWR-1, LY2109761, SB431542 and SB415286 were purchased from Selleckchem (Houston, TX).

Gao F., Alwhaibi A., Sabbineni H., Verma A., Eldahshan W, & Somanath P.R. (2017). Suppression of Akt1- β-catenin pathway in advanced prostate cancer promotes TGFβ1-mediated epithelial to mesenchymal transition and metastasis. Cancer letters, 402, 177-189.

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Signaling Pathways Modulate Gap27 Gene Expression

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To determine the role of key signaling pathways in Gap27-induced gene expression, we blocked TGF-β pathway with SB431542 (20 μM; Selleckchem, Houston, TX, USA), MEK1/2 with PD184352 (10 μM; Sigma-Aldrich), p38 with SB203580 (10 μM; Cell Signaling, Danvers, MA, USA), GSK3α/β with SB415286 (30 μM; Biomol, Hamburg, Germany), AP1 with curcumin (30 μM; Sigma-Aldrich), and SP1 with WP631 (5 nM; Sigma-Aldrich) in Gap27-treated cells, respectively. To this end, confluent GFBL cultures were pre-incubated with inhibitors at 37°C for 1 h, and then treated with Gap27 (150 μM) with or without the inhibitors in serum-free growth medium for 24 h. All inhibitors were dissolved in DMSO, and control samples were treated with respective amounts of DMSO only. Total RNA was collected for real-time PCR as described above.

Tarzemany R., Jiang G., Larjava H, & Häkkinen L. (2015). Expression and Function of Connexin 43 in Human Gingival Wound Healing and Fibroblasts. PLoS ONE, 10(1), e0115524.

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Smad Signaling in Osteo-/Odonto-genesis

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Following 12-hr serum starvation, hBMSCs were cultured in osteo-/odonto-genesis induction medium (OIM) with or without 0.1, 1, 10 and 50 ng/mL rhTGFβ1, pH7.4 or pH10 dentin extracts, 700 ng/mL Noggin (R&D Systems) or 10 μM SB431542 (Selleckchem). After 30-min or 7-day culture, cells were washed with ice-cold PBS followed by protein extraction in RIPA Lysis Buffer (Thermo Scientific) with Protease/Phosphatase Inhibitor Cocktails (Cell Signaling Technology). Proteins were separated on a NuPAGE® Novex® 4–12% Bis-Tris Protein Gel (1.0 mm), transferred to nitrocellulose membrane (Bio-Rad), and detected with anti-phospho-Smad 2/3 (ab52903, 1:500, Abcam), anti-Smad 2/3 (ab202445, 1:500, Abcam), anti-Smad 1/5/9 (ab66737, 1:500, Abcam), anti-phospho Smad 1/5/8 (ab3848, 1:500, Millipore), anti-COL-1 (ab34710, 1:500, Abcam), anti-DSPP (sc-73632, 1:200, Santa Cruz Biotechnology), anti-RUNX2 (ab76956, 1:500, Abcam), anti-GAPDH (sc-25778, 1:200, Santa Cruz Biotechnology) antibodies. Images were developed with IR fluorescence & Odyssey using corresponding secondary antibodies (LI-COR).

Wang S., Niu Y., Jia P., Liao Z., Guo W., Chaves R.C., Tran-Ba K.H., He L., Bai H., Sia S., Kaufman L.J., Wang X., Zhou Y., Dong Y, & Mao J.J. (2021). Alkaline activation of endogenous latent TGFβ1 by an injectable hydrogel directs cell homing for in situ complex tissue regeneration. Bioactive Materials, 15, 316-329.

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Efficient hPGCLC Induction from hESCs

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Induction of hPGCLCs was performed according to a previous report [26 (link)]. Briefly, hESCs were dissociated with 0.5 mM EDTA/PBS and 1 × 10⁶ cells per well were plated on Matrigel coated 6-well plates in GK15 medium (G-MEM [Thermo Fisher, 11,710–035], 15% KSR [Thermo Fisher, 10,828–028], 0.1 mM NEAA [Thermo Fisher, 11,140–050], 2 mM L-glutamine [Thermo Fisher, 35,050–061], 1 mM sodium pyruvate [Thermo Fisher, 11,360–070], 0.1 mM 2-mercaptoethanol [Sigma, M3148], 3 μM CHIR99021 [Selleck Chemicals, S2745], 50 ng/ml Activin A [PEPRO TECH, 120-14E] and 10 μM ROCK inhibitor) for pre-induction. After 40 ~ 42 h of pre-induction, the cells were dissociated with Accutase (Thermo Fisher, A1110501) and plated into ultra-low cell attachment U-bottom 96-well plates (Corning, 7007) at a density of 2,000–4,000 cells per well to form embryoid bodies in 200 μl of aRB27 induction medium (Advanced RPMI 1640 [Thermo Fisher, 12,633–012], 1% B-27 supplement [Thermo Fisher, 17,504–044], 0.1 mM NEAA, 2 mM L-glutamine, 500 ng/ml BMP4 (R&D Systems, 314-BP-050), 10 ng/ml human LIF (R&D Systems, 7734-LF-100), 100 ng/ml SCF (R&D Systems, 255-SC-050), 50 ng/ml EGF (R&D Systems, 236-EG-200), and 10 μM ROCK inhibitor (Selleck, S1049) or 10 μM SB431542 (Selleck, S1067).

Li Z., Fang F., Long Y., Zhao Q., Wang X., Ye Z., Meng T., Gu X., Xiang W., Xiong C, & Li H. (2022). The balance between NANOG and SOX17 mediated by TET proteins regulates specification of human primordial germ cell fate. Cell & Bioscience, 12, 181.

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Chemical Reagents for Signaling Pathways

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Chemical reagents U0126 (Cell Signaling Technology, #9903), PD98059 (Cell Signaling Technology, #9900), Y-27632 (Cell Signaling Technology, #13624), SB431542 (Selleckchem, S1067), CCG1423 (Selleckchem, S7719), verteporfin (Sigma, SML0534). VS-6064 (Selleckchem, S7654), PF431396 (Selleckchem, S7644), PF562271 (Selleckchem, S2890), and Vanadate (New England Biolabs, P0758) were used at doses and times indicated in figure legends. All compounds were dissolved in DMSO.

Sato T., Verma S., Andrade C.D., Omeara M., Campbell N., Wang J.S., Cetinbas M., Lang A., Ausk B.J., Brooks D.J., Sadreyev R.I., Kronenberg H.M., Lagares D., Uda Y., Pajevic P.D., Bouxsein M.L., Gross T.S, & Wein M.N. (2020). A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction. Nature Communications, 11, 3282.

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Platelet Releasate Modulates T Cell Function

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CD4⁺ and CD8⁺ T cells were purified using magnetic beads to a purity of ≥95%. 1×10⁵ cells were cultured in 96-well plates pre-coated with anti-CD3ε antibody (3 μg/mL), in the presence of IL-2 (100 U/mL) and soluble anti-CD28 (2 μg/mL), together with either media or platelet releasate. On day 3 of culture, T cells were stimulated with PMA (300 ng/mL)/Ionomycin (1 μg/mL) or hgp100 peptide 25–33 (5 μg/mL) for 4 hours in the presence of GolgiPlug (BD Biosciences), followed by staining for relevant markers. TGFβ receptor signaling was blocked using the combination of an ALK5 inhibitor (SB431542, Selleckchem) at 20 μM and anti-TGFβ antibody (R&D Systems, clone MAB1835) at 2 μg/mL. Lactic acid activity was inhibited by blocking the monocarboxylate transporter using α-cyano-4-hydroxycinnamic acid (Sigma, C2020). For CFSE dilution assays, cells were labeled with 5 μM CFSE for 10 minutes at room temperature prior to culture on day 0. Flow cytometry was then performed and the data was analyzed and displayed with FlowJo software. Suppression index by platelet releasate is calculated as: percentage of undivided cells treated with a given fraction of platelet releasate/percentage of undivided cells in the control media.

Rachidi S., Metelli A., Riesenberg B., Wu B.X., Nelson M.H., Fugle C.W., Paulos C.M., Rubinstein M.P., Garrett-Mayer E., Hennig M., Bearden D.W., Yang Y., Liu B, & Li Z. (2017). Platelets Subvert T Cell Immunity Against Cancer via GARP-TGFβ Axis. Science immunology, 2(11), eaai7911.

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