Human peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll density gradient using Ficoll Plaque Premium (Sigma GE Healthcare, #17–544-02) or BD Vacutainer CPT Tubes (BD, #362753) as described somewhere else. PBMC were washed twice with HBSS (Gibco ThermoFisher, #14175095) containing 2% BSA, 1 mm EDTA. Monocytes were obtained by negative selection using a kit (StemCell, #19359). Monocytes were incubated with RPMI 1640 (Sigma Aldrich, #10–040) medium containing 10% de-complemented FBS (OmegaScientific, #FB-02), 1 mM Sodium Pyruvate (Gibco Thermofisher), Penicillin-Streptomycin (15140122, 1000 U/mL) and 25 ng/mL-50 ng/mL of recombinant human M-CSF (StemCell, #78057–2) for six days at 37°C and 5% CO2. Fresh media was added 48 h and four days after seeding. The mDM were incubated for five hours with 100 ng/mL of Kdo2 lipid KLA or 250 μg/mL of MSUc in RPMI 1640 medium containing 10% de-complemented FBS, 1 mM Sodium Pyruvate, Penicillin-Streptomycin and 25ng–50 ng/mL of recombinant human M-CSF. 20 μM of JNK inhibitor SP600125 (Tocris, #1496) was added to the media one hour prior treatment with MSUc.
Jnk inhibitor sp600125
Manufactured by Bio-Techne
Sourced in United States
JNK inhibitor SP600125 is a potent and selective inhibitor of the c-Jun N-terminal kinase (JNK) pathway. It acts by blocking the enzymatic activity of JNK, a key signaling molecule involved in various cellular processes. The compound is commonly used in research applications to study the role of the JNK pathway in biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Vacutainer cpt tube, by BD (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
De complemented fbs, by Omega Scientific (1 mentions)
Penicillin streptomycin, by STEMCELL (1 mentions)
Bay 11 7082, by Bio-Techne (1 mentions)
Erk inhibitor u0126, by Cell Signaling Technology (1 mentions)
Il 1ra, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 1α, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
Mmp 9, by Merck Group (1 mentions)
Mek inhibitor u0126, by Cell Signaling Technology (1 mentions)
Matrigel basement membrane matrix, by BD (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Takara Bio (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
6 protocols using jnk inhibitor sp600125
1
Differentiation and Stimulation of Human Monocyte-Derived Macrophages
2
Investigating Cytokine Signaling in Human Mesangial Cells
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Human mesangial cells (HMC)48 (link) were grown in DMEM containing 10% FCS, 100 units/ml penicillin, and 100 mg/ml streptomycin. Recombinant human IL-1α, IL-1β, IL-1Ra, human CXCL1, 2, 8 ELISA kits were purchased from PeproTech (Rocky Hill, NJ, USA). Mouse anti-human IL-1R1 antibody was purchased from Abcam (Cambridge, UK). Rabbit anti-human phosphorylated ERK, JNK, and p38 antibodies, rabbit anti-human A20 antibody, and ERK inhibitor U0126 were purchased from Cell Signaling Technology (Beverly, MA, USA). P38 inhibitor SB203580, JNK inhibitor SP600125, and NF-κB inhibitor Bay117082 were purchased from Tocris (Ellisville, MO, USA). A20 mammalian expressing plasmid (EX-K6040-M11) was purchased from Genecopoeia (Rockville, MD, USA).
3
Molecular Mechanism of BRA-90 Anthocyanin Effects
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BRA-90 anthocyanins (BRACs) extract was purchased from New Star (Jilin, China). Antibodies against total HER2, total K-RAS, total extracellular signal-regulated kinase 1/2 (ERK1/2), total RAF1, and phosphorylated (p)-RAF1 (Ser259) were obtained from Abcam (Cambridge, UK). Antibodies against total mitogen-activated protein kinase 1 (MEK1), p-MEK1/2 (Ser218/222), p-ERK (Thr202/Tyr204), total c-Jun N-terminal kinase 1/2 (JNK1/2), p-JNK1/2 (Thr183/Tyr185), matrix metalloproteinase (MMP2), and MMP9 were obtained from Millipore (Billerica, MA, USA). Polyethylene terephthalate membrane (8 μm pore size) Millicell hanging cell culture inserts were purchased from Millipore. BD Matrigel Basement Membrane Matrix was obtained from Becton and Dickinson (Franklin Lakes, NJ, USA). The RAF inhibitor (ZM336372) was purchased from Santa Cruz Biotech (Dallas, TX, USA), MEK inhibitor (U0126) was purchased from Cell Signaling Technology (Danvers, MA, USA), and JNK inhibitor (SP600125) was purchased from Tocris (Bristol, UK). The SYBR Select Master Mix (category number 4472908), raf, mek, jnk, and glyceraldehyde 3-phosphate dehydrogenase (gapdh) universal primers, Trizol Reagent, and Lipofectamine 3000 were purchased from Invitrogen (Carlsbad, CA, USA), and the cDNA Synthesis Kit was purchased from TaKaRa Bio (Otsu, Japan).
4
Modulation of Fibroblast Responses
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rhIL-17A, rhTGF-β, monoclonal mouse IgG1 TGF-β1, 2, 3 antibody, IL-6, MCP-1, MMP-1, IL-8, pro-collagen Iα1 and fibronectin ELISA DuoSet kits were from R&D Systems (Abingdon, UK); DMEM, PBS, glutamine, penicillin, streptomycin, trypsin, dispase, collagenase type I from Gibco (Paisley, UK); FCS from Biowest (Nuaillé, France); BSA, α-ketoglutaric acid, β-amino propionitrile, l -ascorbic acid, p38 MAPK inhibitor SB203580, and PI3K inhibitor LY294002 from Sigma (St. Louis, MO, USA); MEK1/2 inhibitor U-0126 from Calbiochem (San Diego, CA, USA); TGF-βRI inhibitor SD 208, JNK inhibitor SP 600125 and IKK-2 inhibitor TPCA-1 from Tocris Bioscience (Bristol, UK); LEAF irrelevant control mAbs from Biolegend (San Diego, CA, USA); Complete Protease Inhibitor Cocktail and PhosSTOP phosphatase inhibitor from Roche (Basel, Switzerland); nitrocellulose membranes and chemiluminescence (ECL) blotting analysis system from GE Healthcare (Zurich, Switzerland); phospho-Akt (Ser473), phospho-Smad2 (Ser465/467), phospho-p38 MAPK (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), phospho-IκB-α (Ser32), β-actin and BSA for Western blots from Cell Signaling (Danvers, MA, USA); TMB ELISA substrate from Abcam (Cambridge, UK); EZ4U cell proliferation assay from Biomedica (Vienna, Austria).
5
Protective Effects of Berberine on Visfatin-Induced Endothelial Injury
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HUVECs (Cascade Biologics, Portland, OR, USA) were cultured in endothelial cells basal medium supplemented with 10% fetal bovine serum (FBS; Invitrogen), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere containing 5% CO2 and grown to 70–80% confluence before being treated with the indicated agents. Cells between passages 3 and 7 were used in all experiments. To further elucidate the protective effect and the potential mechanism of BBR on visfatin-induced HUVECs injury, HUVECs were pretreated with BBR (50 µmol/l; Sigma), p38 MAPK inhibitor SB203580 (20 µmol/l) or JNK inhibitor SP600125 (10 µmol/l) (both from Tocris Bioscience, Ellisville, MO, USA) for 1 h and followed by the addition of human recombinant visfatin (100 ng/ml; Peprotech) for 24 h.
6
Signaling Pathway Modulation in Inflammation
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Rabbit anti-human A20 and GAPDH antibodies, rabbit anti-human phosphorylated ERK, JNK, p38, and NF-κB P65 antibodies, and the ERK inhibitor U0126, were purchased from Cell Signaling Technology (Beverly, MA, USA). The NOD2 ligand MDP was purchased from Invivogen (San Diego, CA, USA). The TLR4 ligand lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Human IL-8 and IL-1β ELISA kits were purchased from Jiamay Biotech. (Beijing, China). The A20 expression plasmid was purchased from GeneCopoeia (Germantown, MD, USA). The JNK inhibitor SP600125 and P38 inhibitor SB202190 were purchased from Tocris (Bristol, UK). The mouse anti-human NOD2 antibody and NF-κB inhibitor Bay 11-7082 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant human IL-1α and IL-1β were purchased from PeproTech Inc. (Rocky Hill, NJ, USA). NF-κB inhibitor BAY 11-7085 was purchased from MedChemExpress (Monmouth Junction, NJ, USA).
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