Sp600125

Manufactured by Cell Signaling Technology

Sourced in United States, Germany

SP600125 is a selective, cell-permeable inhibitor of the c-Jun N-terminal kinase (JNK) signaling pathway. It binds to and inhibits the catalytic activity of JNK1, JNK2, and JNK3 enzymes.

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JNK inhibitor SP600125 (12 citations)

101 protocols using sp600125

Modeling Astrocyte Responses to Oxygen-Glucose Deprivation

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An oxygen-glucose deprivation and reoxygenation (OGD/R) model was constructed as previously described (19 (link)). Having being washed twice, astrocytes were immersed in glucose-free DMEM and placed in an incubator with a premixed gas (1% O₂, 95% N₂ and 5% CO₂) for 4 h. Then, cells were maintained in normal DMEM (including 5.6 mmol/l glucose) and transferred to a 5% CO₂ incubator at 37 °C for 24 h. For the non-OGD/R group, cultures were incubated in normal DMEM and placed in 5% CO₂ in air at 37°C for 28 h. Astrocytes were also treated with Ad-GFP, Ad-BMP9, Ad-BMP9 + extracellular signal-regulated kinases (ERK) inhibitor PD098059 (20 µM; Sigma-Aldrich; Merck KGaA), Ad-BMP9 + c-Jun N-terminal kinase (JNK) inhibitor SP600125 (20 µm; Cell Signaling Technology, Inc., Danvers, MA, USA), Ad-BMP9 + p38 inhibitor SB203580 (20 µm; Cell Signaling Technology, Inc.), PD098059 (20 µm), SP600125 (20 µm), and SB203580 (20 µm) for 24 h and then subjected to OGD/R treatment.

Feng Y, & Hu Y. (2017). Bone morphogenetic protein 9 serves a protective role in response to ischemic-reperfusion in the brain by promoting ERK activation. Molecular Medicine Reports, 17(2), 2845-2852.

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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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Magnolol Regulates Lipid Metabolism

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Magnolol, purity ≥98%, was purchased from Chengdu Reference Products (Chengdu, China). Dimethylsulfoxide (DMSO), oleic acid (OA), tyloxapol (Ty), and Oil Red O were obtained from Sigma-Aldrich (St. Louis, MO, USA). An Oil Red O stain kit (for cultured cells) was purchased from Solarbio Science & Technology (Beijing, China). Reactive oxygen species (ROS), TG, and total cholesterol (TC) assay kits were provided by Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). Human TNF-α enzyme-linked immunosorbent assay (ELISA) kits were provided by BioLegend (CA, USA). U0126, SB203580, SP600125, and LY290042 (specific inhibitors of ERK1/2, P38, JNK1/2, and AKT, respectively) and antibodies against AMPKα, P-AMPKα, AMPKβ, P-AMPKβ, adenosine ACC, P-ACC, P-ERK1/2, ERK1/2, P-JNK1/2, JNK1/2, P-P38, P38, IκB, P-IκB, and P-P65 were purchased from Cell Signaling Technology (Boston, MA, USA). AKT, P-AKT (Thr 308) and GAPDH antibodies were purchased from Affinity (OH, USA). Antibodies against P65, SREBP-1c and β-actin were purchased from Proteintech (Boston, MA, USA). PPARα and compound c (inhibitor of AMPK) were purchased from Abcam (Cambridge, MA, USA). HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies were provided by Boster (CA, USA). All other chemicals were of reagent grade.

Tian Y., Feng H., Han L., Wu L., Lv H., Shen B., Li Z., Zhang Q, & Liu G. (2018). Magnolol Alleviates Inflammatory Responses and Lipid Accumulation by AMP-Activated Protein Kinase-Dependent Peroxisome Proliferator-Activated Receptor α Activation. Frontiers in Immunology, 9, 147.

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Inflammasome Activation and Inhibition Assays

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MCC950 was purchased from TargetMol (Wellesley Hills, MA). Phorbol myristate acetate (PMA), ammonium chloride (NH₄Cl), chloroquine diphosphate (CQ), N-acetyl-cysteine (NAC), pyrrolidine dithiocarbamate (PDTC), potassium chloride (KCl), glybenclamide, probenecid, carbenoxolone and cyclosporine A were purchased from Sigma-Aldrich (St. Louis, MO). Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) and antibodies against ASC, IL-18, TLR4, P₂X₇, and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). PD98059, SB203580, SP600125, and antibodies against phospho-extracellular signal-regulated kinases 1/2 (ERK1/2), phospho-c-Jun N-terminal kinases 1/2 (JNK1/2), phospho-p38 and human caspase-1 were obtained from Cell Signaling Technology (Beverly, MA). Antibodies against NLRP3 and mouse caspase-1 were purchased from Adipogen International (San Diego, CA). Antibodies against IL-1β were purchased from R&D Systems (Minneapolis, MN). 10panx was purchased from Tocris Bioscience (Bristol, UK). The pHrodo Green E. coli BioParticles Conjugate was purchased from Thermo Fisher Scientific (Waltham, MA).

Li L.H., Lin J.S., Chiu H.W., Lin W.Y., Ju T.C., Chen F.H., Chernikov O.V., Liu M.L., Chang J.C., Hsu C.H., Chen A., Ka S.M., Gao H.W, & Hua K.F. (2019). Mechanistic Insight Into the Activation of the NLRP3 Inflammasome by Neisseria gonorrhoeae in Macrophages. Frontiers in Immunology, 10, 1815.

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Generation and Use of Polyclonal Antibodies

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A custom p-FOXO3 rabbit polyclonal antibody was generated as described previously.^{12 (link)} Anti-HA antibody (ab9110) was purchased from Abcam (Cambridge, MA, USA). Anti-FOXO3 (75D8), anti-acetylated-lysine (9441), anti-Bcl-2 (50D3), anti-TRAIL (C92B9), anti-SIRT1 (D1D7), anti-SIRT7 (D3K5A), anti-SAPK/JNK, p-SAPK/JNK (T183/Y185) and anti-poly (ADP-ribose) polymerase (9542) were purchased from Cell Signaling Technology (Boston, MA, USA). Anti-SIRT6 was purchased from Abnova (Walnut, CA, USA). Anti-GAPDH (FL-335) and anti-TRAIL (H-257) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-actin (AC-15), anti-SIRT2 and anti-Flag (M2) were purchased from Sigma-Aldrich (St Louis, MO, USA). RSV, TSA, C646 and sirtinol were purchased from Sigma-Aldrich. NAD⁺ and CAY10591 were purchased from Cayman Chemical (Ann Arbor, MI, USA). rSIRT7 was purchased from SignalChem (Richmond, BC, Canada). SP600125 was purchased from Cell Signaling Technology. SB203580 was purchased from AdipoGen (San Diego, CA, USA). FR180204 was purchased from Calbiochem (Billerica, MA, USA). LPS from Escherichia coli O55:B5 was purchased from Enzo Life Science (Farmingdale, NY, USA).

Li Z., Bridges B., Olson J, & Weinman S. (2016). The interaction between acetylation and serine-574 phosphorylation regulates the apoptotic function of FOXO3. Oncogene, 36(13), 1887-1898.

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Kinase Inhibitor Pretreatment for PAMP Stimulation

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Inhibitors specific for JNK (SP600125), MEK1/2 (U0126, to prevent ERK activity), and p38 (SB203580) were purchased from Cell Signaling Technology. Cells were first treated with 10 μM inhibitors for 50 min, which was followed by stimulation with the appropriate PAMP. The cell culture medium was not changed after the cells were treated with inhibitor.

Liu B., Liu Q., Yang L., Palaniappan S.K., Bahar I., Thiagarajan P.S, & Ding J.L. (2016). Innate immune memory and homeostasis may be conferred through crosstalk between the TLR3 and TLR7 pathways. Science signaling, 9(436), ra70.

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Inhibition of Signaling Pathways in DCs

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DCs were pretreated with vehicle (0.1% DMSO) or selective inhibitors for ERK1/2 (U0126; 10, 50, and 100 µM), JNK (SP600125; 1, 5, and 10 µM), p38 (SB203580; 5, 25, and 50 µM), and NF-κB (BAY1170-82; 10, 100, and 500 µM) (all from Cell Signaling Technology, Danvers, MA, USA) for 45 min. Subsequently, cells were stimulated with B. abortus S2308 strain (MOI 100:1) or with bacterial RNA (2 µg/mL) for 24 h, and harvested supernatants were used to measure cytokine production by ELISA.

Campos P.C., Gomes M.T., Guimarães E.S., Guimarães G, & Oliveira S.C. (2017). TLR7 and TLR3 Sense Brucella abortus RNA to Induce Proinflammatory Cytokine Production but They Are Dispensable for Host Control of Infection. Frontiers in Immunology, 8, 28.

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Viability Assay of Cell Lines

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The viability of the cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Sigma-Aldrich, St. Louis, MO, USA). The 2 × 10⁴–4 × 10⁴ cells/mL were seeded in 96-well plates. After the attachment, cells were treated with different concentrations of B, EL000327, D, and 5-FU for 48 h in the presence or absence of various inhibitors (Z-VAD-FMK (10 µM) (R&D System, Inc, McKinley Place N.E, MN, USA), 3-MA (1mM), CQ (10 µM), NAC (5 mM) (Sigma-Aldrich, St. Louis, MO, USA), and SP600125 (10 µM) (Cell Signaling Technology, MA, USA). Cells treated with 0.01% of DMSO were used as the control. Next, 15 µL of the MTT reagent was added to each well and incubated for 4 h at 37 °C. The medium was aspirated completely and 150 µL of DMSO (Sigma-Aldrich, St. Louis, MO, USA) was added to the cells before the absorbance was measured at 540 nm by a microplate reader (Bio Tek Instruments, Winooskim, VT, USA) using Gen 5 (2.03.1) software. SPSS statistical software 23 was used for the IC₅₀ calculation. Synergic effects of B with D or 5-FU were assessed by compuSyn software.

Gamage C.D., Kim J.H., Yang Y., Taş İ., Park S.Y., Zhou R., Pulat S., Varlı M., Hur J.S., Nam S.J, & Kim H. (2023). Libertellenone T, a Novel Compound Isolated from Endolichenic Fungus, Induces G2/M Phase Arrest, Apoptosis, and Autophagy by Activating the ROS/JNK Pathway in Colorectal Cancer Cells. Cancers, 15(2), 489.

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Studying NF-κB Activation Mechanisms

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To study the action mechanism, various pharmacological inhibitors were tested on PMA-mediated NF-κB activation. Pharmacological inhibitors were used such as Wortmannin, Bay 11-7082, Genistein, GF109203X, PD98059, SB203580, SP600125, and U-73122 (Cell Signaling Technology, Danvers, MA) for the inhibition of phosphoinositide 3-kinase (PI3K), IkB-α phosphorylation, protein tyrosine kinase (PTK), protein kinase C (PKC), MEK1, SAPK2 (p38), jun N-terminal kinase (JNK), and phospholipase C (PLC), respectively.

Oh D.R., Kang H.W., Kim J.R., Kim S., Park I.K., Rhee J.H., Oh W.K, & Kim Y.R. (2014). PMA Induces Vaccine Adjuvant Activity by the Modulation of TLR Signaling Pathway. Mediators of Inflammation, 2014, 406514.

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Thyroid Cancer Cell Lines and Inhibitors

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The K1 (papillary cancer), FTC-133 (follicular cancer), and 8505C (anaplastic cancer) thyroid cancer cell lines were from the European Collection of Cell Cultures (ECACC, Salisbury, United Kingdom). Cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Life Technology, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY) in a humidified cell culture incubator at 37 °C and 5% CO₂ . PD98059 (ERK1/2 inhibitor), SP600125 (JNK inhibitor), and SB203580 (p38 inhibitor) were purchased from Cell Signaling Technology (Beverly, MA).

Guan H., Guo Z., Liang W., Li H., Wei G., Xu L., Xiao H, & Li Y. (2017). Trop2 enhances invasion of thyroid cancer by inducing MMP2 through ERK and JNK pathways. BMC Cancer, 17, 486.

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