Sb431542

Manufactured by Abcam

Sourced in United States

SB431542 is a selective and potent inhibitor of the transforming growth factor-beta (TGF-β) type I activin receptor-like kinase (ALK) receptors ALK4, ALK5 and ALK7. It inhibits the phosphorylation and activation of SMAD2 and SMAD3, which are key mediators of the TGF-β signaling pathway.

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Lab products found in correlation

37 protocols using sb431542

iPSC-Derived Neural Progenitor Differentiation

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Chinese hamster ovary (CHO) control H1 and NPC1-null M12 cells8 (link) were grown as monolayers at 37°C/5%
CO₂ in Dulbecco’s Modified Eagle’s Medium
(DMEM)/F-12 (Thermofisher) with 1% L-glutamine (Lonza), 10% heat-inactivated
foetal bovine serum (Sigma/Pan Biotech) either with or without HEPES/other
zwitterionic buffer at pH 7.4 (Thermofisher/Lonza).
Control induced pluripotent stem cell (iPSC)-derived neural progenitor
cells (NPCs) were cultured on vitronectin-coated 6-well plates with E8 flex
medium (Life Technologies) at 37 °C/5% CO₂. Neural induction
proceeded according to previous methods9 (link)
with modifications. Briefly, NPCs were derived in Advanced DMEM/F-12 (ADF) with
GlutaMAX, penicillin/streptomycin (Life Technologies), 2% NeuroBrew 21 without
retinoic acid (Miltenyi), LDN193189 (1 µM, Stemgent), SB431542 (10 µM,
Abcam) and IWR1 (1.5 µM, Tocris). NPCs were expanded in ADF with 2%
NeuroBrew 21 with retinoic acid (Miltenyi Biotec) and 10 ng/mL basic fibroblast
growth factor. NPCs were terminally differentiated in SynaptoJuiceA (HEPES-free)
for 7-days, followed by two weeks in SynaptoJuiceB (5.5 mM HEPES) according
to9 (link),10 (link). Neurons were maintained in SynaptoJuiceB, both with and without
additional 10 mM HEPES for 7 days.

Cook S.R., Badell-Grau R.A., Kirkham E.D., Jones K.M., Kelly B.P., Winston J., Waller-Evans H., Allen N.D, & Lloyd-Evans E. (2020). Detrimental effect of zwitterionic buffers on lysosomal homeostasis in cell lines and iPSC-derived neurons. AMRC open research, 2, 21.

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Tenocyte Differentiation of C2C12 and C3H10T1/2 Cells

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C2C12 and murine mesenchymal cells (C3H10T1/2) were cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% FBS and antibiotics. The cells were passaged before reaching confluence and used within 10 passages. To induce tenocyte differentiation, C2C12 cells were cultured in serum‐free AIM‐V medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with GDFs (500 ng·mL⁻¹ of GDF‐5, ‐6, ‐7 and 10, 100, 500 ng·mL⁻¹ of myostatin; R&D Systems, Minneapolis, MN, USA). The medium was replaced every 2 days. To analyze signaling pathways, cells were treated with 500 ng·mL⁻¹ of myostatin and 10 μmol·L⁻¹ of inhibitors for ALK (SB431542), p38MAPK (SB203580), and MEK1 (PD98059) (Abcam, Cambridge, MA, USA) for 5 days.

Uemura K., Hayashi M., Itsubo T., Oishi A., Iwakawa H., Komatsu M., Uchiyama S, & Kato H. (2017). Myostatin promotes tenogenic differentiation of C2C12 myoblast cells through Smad3. FEBS Open Bio, 7(4), 522-532.

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Directed Differentiation of hiPSCs using Inhibitors

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The following chemicals were added to the EB medium during EB formation of hiPSCs: 10 μM SB431542 (SB, Abcam, Cambridge, MA, USA), 5 μM dorsomorphin (DM, A.G. Scientific), and 10 μM phenylhydrazonopyrazolone sulfonate 1 (PHPS1, Calbiochem, Darmstadt, Germany).
Inhibitor of SHP2 (10 μM PHPS1) was treated in NPCs for 7 days before neural differentiation and the inhibitor also added in cerebral organoids between 28 and 35 days of culture.

Ju Y., Park J.S., Kim D., Kim B., Lee J.H., Nam Y., Yoo H.W., Lee B.H, & Han Y.M. (2020). SHP2 mutations induce precocious gliogenesis of Noonan syndrome-derived iPSCs during neural development in vitro. Stem Cell Research & Therapy, 11, 209.

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Inhibition of Wnt/β-catenin and TGF-β1/Smads Pathways

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AS-IV (Figure 1; C₄₁H₆₈O₁₄; molecular weight =784.97, purity by high-performance liquid chromatography ≥98%) was purchased from Sigma-Aldrich (Milwaukee, WI, USA). The Wnt/β-catenin pathway inhibitor XAV-939 and the TGF-β1/Smads pathway inhibitor SB431542 were purchased from Abcam (Cambridge, UK). Rabbit polyclonal anti-α-SMA, anti-TGF-β1, anti-Smad3 (phospho S423 + S425; P-Smad3), anti-fibronectin (FN), and anti-Collagen IV (Col IV) antibodies were purchased from Abcam. Mouse monoclonal anti-Smad7 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit monoclonal anti-non-phospho β-catenin (activated β-catenin) antibody was ordered from Cell Signaling Technology (Danvers, MA, USA). Rabbit monoclonal anti-nephrin antibody was provided by Novus Biologicals (Littleton, CO, USA). miR-21 mimics and negative controls were synthesized by Ribobio (Guangzhou, China).

Wang X., Gao Y., Tian N., Zou D., Shi Y, & Zhang N. (2018). Astragaloside IV improves renal function and fibrosis via inhibition of miR-21-induced podocyte dedifferentiation and mesangial cell activation in diabetic mice. Drug Design, Development and Therapy, 12, 2431-2442.

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TGF-β Signaling Inhibition in Ectopic Mineralization

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To test the effect of suppressed TGF-β signalling on DC/HO pathogenesis, a TGF-β type I receptor inhibitor, SB431542 (10 mg/kg in corn oil vehicle containing 5% dimethyl sulfoxide (DMSO); Abcam) was administered by daily subcutaneous injection for seven days into eight- to 12-week-old male C57BL/6 J mice that received an injection of 50 μl of 10 μM CTX into the calf muscles at Day 1. Mice were killed at Day 7 for microCT analysis of ectopic mineralization. Mice injected daily with corn oil vehicle containing 5% DMSO combined with CTX treatment served as control.

Li L., Xiang S., Wang B., Lin H., Cao G., Alexander P.G, & Tuan R.S. (2020). Dead muscle tissue promotes dystrophic calcification by lowering circulating TGF-β1 level: implications for post-traumatic heterotopic ossification. Bone & Joint Research, 9(11), 742-750.

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Midbrain Organoid Generation Protocol

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Midbrain organoids generation is described in (Nickels et al., 2020 (link); Smits et al., 2019 (link)). Shortly, 6000 cells per well were seeded in an Ultra‐Low Attachment 96‐well Well plate (Merck, CLS3474) and kept under maintenance conditions (N2B27 medium supplemented with 0.2 mM Ascorbic acid [Sigma, A4544‐100G], 3 μM CHIR 99021 [Axon, CT 99021], 0.5 μM Smoothened Agonist [SAG, Stem cell technologies, 73412], 2.5 μM SB‐431542 [Abcam, ab120163], 0.1 μM LDN‐193189 [Sigma, SML0559]) for 2 days. After that, we started the pre‐patterning (day 0 of dopaminergic differentiation) by removing SB and LDN from the medium. Two days after, CHIR concentration was reduced to 0.7 μM. On day 6 of dopaminergic differentiation, the medium was changed into maturation medium (N2B27 plus 0.2 mM) Ascorbic acid, 10 ng/ml Brain Derived Neurotrophic Factor, BDNF (Peprotech, 450‐02), 10 ng/ml Glial‐Derived Neurotrophic Factor, GDNF (Peprotech, 450‐10), 1 pg/ml TGF‐β3 (Peprotech, 100‐36E), 0.5 mM db cAMP (Sigma, D0627‐5X1G), 10 μM DAPT (R&D Systems, 2634/10) and 2.5 ng/ml Activin A (Thermo Scientific, PHC9564). Organoids were kept under static culture conditions with media changes every third day until day 15 of dopaminergic differentiation.

Sabate‐Soler S., Nickels S.L., Saraiva C., Berger E., Dubonyte U., Barmpa K., Lan Y.J., Kouno T., Jarazo J., Robertson G., Sharif J., Koseki H., Thome C., Shin J.W., Cowley S.A, & Schwamborn J.C. (2022). Microglia integration into human midbrain organoids leads to increased neuronal maturation and functionality. Glia, 70(7), 1267-1288.

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Tumor-Macrophage Interaction Modulation

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DU145 cells (1 × 10⁵) and bone-marrow-derived macrophages (1 × 10⁵) were co-cultured in the system with/without SB431542 (10 µmol/L, Abcam, Cambridge, MA, USA), or with/without recombinant PDGF (150 ng/ml, Sigma-Aldrich, St. Louis, MO, USA), or with/without anti-PDGFR (15 µg/L, R&D Biosystem, Los Angeles, CA, USA) for 2 days. After co-culture, an MTT assay was applied to detect the number of DU145 cells and recorded the number variation. In addition, flow cytometry was applied to assess macrophage subtypes.

Cui F., Xu Z., Hu J, & Lv Y. (2022). Spindle pole body component 25 and platelet-derived growth factor mediate crosstalk between tumor-associated macrophages and prostate cancer cells. Frontiers in Immunology, 13, 907636.

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Neural Induction and Differentiation

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Neural induction was performed as previously reported³³. Briefly, cells were dissociated to single cells using Accutase and plated on gelatin for 10 minutes to remove MEFs. Non-adherent cells were collected and plated on Geltrex-treated dishes at a density of 150-200k cells per well of a 24-well plate in the presence of MEF-conditioned hESC media containing 10 ng/ml of FGF-2 (Life Tech) and 10 uM of Y-27632 (Tocris). Neural differentiation was initiated when cells were confluent using KSR media containing 820 ml of Knockout DMEM (Life Tech), 150 ml Knockout Serum Replacement (Life Tech), 1 mM L-glutamine (Life Tech), 100 uM MEM non-essential amino acids (Life Tech), and 0.1 mM beta- mercaptoethanol (Life Tech) to inhibit SMAD signaling, 100 nM of LDN-193189 (Cat. no. ab142186, Abcam) and 5 uM of SB431542 (Cat. No. 13031, Cayman Chemical) were added on Days 0 through 9. Cells were fed daily, and N2 media (Life Tech) was added in increasing 25% increments every other day starting on Day 4 (100% N2 on Day 10).

Choi J., Lee S., Clement K., Mallard W., Tagliazucchi G.M., Lim H., Choi I.Y., Ferrari F., Tsankov A., Pop R., Lee G., Rinn J., Meissner A., Park P.J, & Hochedlinger K. (2015). A comparison of genetically matched cell lines reveals the equivalence of human iPSCs and ESCs. Nature biotechnology, 33(11), 1173-1181.

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Fibroblast Reprogramming via Doxycycline-Inducible dCas9 and gRNAs

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Before initiating reprogramming, human neonatal fibroblast cells (FTC0007) were transduced as described above with three lentiviruses: Tre3G, TetOn-dCas9-10XGCN4-P2A-mCherry and ScFV-GCN4-VP64-GFP. Transduced cells were treated with 1 μg/ml doxycycline for 24 h and were sorted in bulk using fluorescence-activated cell sorting (FACS) for expression of GFP (VP64) and mCherry (dCas9). Sorted fibroblast cells were again transduced with lentiviruses expressing indicated gRNAs. To initiate reprogramming, fibroblast medium supplemented with 1 μg/ml doxycycline was added to induce dCas9 expression. After 1 week, doxycycline treatment was discontinued, and the cells were passaged using Accutase and were seeded onto 0.1% gelatin-coated six-well plates in indicated reprogramming media with or without small molecules PD0325901 (0.4 μM, Abcam), CHIR99021 (1 μM, Abcam), thiazovivin (5 μM, Ryss Labs) and SB431542 (2 μM, Abcam). On week 2, cells were passaged and seeded onto Matrigel-coated six-well plates in iPSC media with or without small molecules PD0325901, CHIR99021 and Thiazovivin. iPSC medium is no longer supplemented with SB431542 at this stage. iPSC colonies were ready to sort by FACS on week 3.

Abujarour R., Dinella J., Pribadi M., Fong L.K., Denholtz M., Gutierrez A., Haynes M., Mahmood E., Lee T.T., Ding S, & Valamehr B. (2024). A chemical approach facilitates CRISPRa-only human iPSC generation and minimizes the number of targeted loci required. Future Science OA, 10(1), FSO964.

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Osteogenesis of Diabetic BMSCs

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BMSCs were induced for osteogenesis following our previously published protocol 28. Briefly, cells were trypsinized, seeded at a density of 5,000 cells per cm², and induced for osteogenesis by medium composed of low‐glucose DMEM, 10% FBS, 1% penicillin and streptomycin, 10 mM β‐glycerophosphate, 50 μg/ml μM l‐ascorbic acid‐2‐phosphate, 0.1 μM dexamethasone, and 10 nM 1α,25‐dihydroxyvitamin D3 (Sigma–Aldrich, St. Louis, MO). The induction medium was changed every 3 days.
To determine if TGFB and/or bone morphogenetic protein (BMP) signaling involved in the regulation of osteogenesis of BMSCs are affected by diabetes, the cell from T1DM (T1DM‐BMSCs) and non‐T1DM donors (non‐T1DM‐BMSCs) were treated with 5 μM of dorsomorphin, an inhibitor of BMP type 1 receptor kinase, and/or 5 μM of SB431542, an inhibitor of TGFB receptor kinase (Abcam, Cambridge, MA) during 21‐day osteogenic differentiation.

Wang J.F., Lee M., Tsai T., Leiferman E.M., Trask D.J., Squire M.W, & Li W. (2019). Bone Morphogenetic Protein‐6 Attenuates Type 1 Diabetes Mellitus‐Associated Bone Loss. Stem Cells Translational Medicine, 8(6), 522-534.

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