BV-2 microglial cells were stimulated for 12 h with 166 µg/ml RAGE-blocking antibody (Abcam, Cambridge, UK), 1.0 µg/ml C225 (inhibitor of TLR4), 20 µM PD98059 [inhibitor of extracellular signaling kinase (ERK)], 7 µM SB203580 (inhibitor of p38 MAP kinase), 10 µM SP600125 (inhibitor of JNK) or 50 µM PDTC (inhibitor of p65 NF-κB) (all from R&D Systems, ON, Canada) for 1 h before addition of 0.1 µM recombinant S100A8/A9 proteins or 1 µg/ml LPS (Escherichia coli O26:B6; Sigma-Aldrich, St. Louis, MO, USA) in the presence of 25 µg/ml polymyxin B (R&D Systems), and the culture supernatants were harvested. Levels of IL-6 and TNF-α in 100 µl medium were measured by commercial ELISA kits (Boster Biological Technology, Wuhan, China) according to the manufacturer's instructions.
Pd98059
Manufactured by R&D Systems
Sourced in Germany
PD98059 is a small molecule inhibitor. It specifically and potently inhibits the activation of the extracellular signal-regulated kinase (ERK) pathway by blocking the activation of MEK1 and MEK2.
Automatically generated - may contain errors
Lab products found in correlation
Fetal calf serum (fcs), by PAN Biotech (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Sp600125, by R&D Systems (1 mentions)
Sb203580, by R&D Systems (1 mentions)
Lps escherichia coli o26 b6, by Merck Group (1 mentions)
Glycine, by AppliChem (1 mentions)
Sodium dodecyl sulphate (sds), by AppliChem (1 mentions)
Glycerol, by AppliChem (1 mentions)
Orthovanadate, by Merck Group (1 mentions)
Milk powder, by Bio-Rad (1 mentions)
Tween 20, by Carl Roth (1 mentions)
Mgcl2, by AppliChem (1 mentions)
Hepes, by AppliChem (1 mentions)
Phenylmethylsulfonyl fluoride pmsf, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Luminol, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Capsazepine, by Merck Group (1 mentions)
7 protocols using pd98059
1
Microglial Inflammatory Signaling Modulation
2
Cannabinoid Receptor Signaling Pathway
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THC was obtained from Lipomed (Weil am Rhein, Germany). O-1602 and PD98059 were bought from R&D Systems (Wiesbaden-Nordenstadt, Germany). AM-251 and AM-630 were purchased from Biomol GmbH (Hamburg, Germany). Dulbecco's Modified Eagle's medium (DMEM, high glucose, GlutaMAXTM), trypsin-EDTA and penicillin-streptomycin were obtained from Life Technologies GmbH (Darmstadt, Germany). Fetal calf serum (FCS) was from PAN Biotech (Aidenbach, Germany). Dimethyl sulfoxide (DMSO), EDTA, glycerol, glycine, H2O2, HEPES, MgCl2, NaCl, sodium dodecyl sulphate and Tris were bought from AppliChem GmbH (Darmstadt, Germany). Aprotinin, capsazepine, HCl, leupeptin, luminol, orthovanadate, para-cumaric acid, phenylmethylsulfonyl fluoride (PMSF) and Triton® X-100 were obtained from Sigma-Aldrich (Taufkirchen, Germany). Milk powder was obtained from Bio-Rad Laboratories GmbH (Munich, Germany). Tween® 20 was purchased by Carl Roth GmbH (Karlsruhe, Germany).
3
Metastatic Breast Cancer Cell Lines
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Human breast cancer cell lines MDA-MB-231 and Hs578T were obtained and cultured as recommended by American Type Culture Collection (ATCC; Gaithersburg, MD, USA). Characterization of MDA-MB-231/B02 (MDA-MB/B02) and MDA-BO2.Luc breast cancer cells and culture conditions were described previously [40 (link)]. Lysophosphatidic acid (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate; 18:1) was obtained from Avanti Polar Lipids Inc, Alabama, USA. Ki16425 (LPA1/LPA3 antagonist) was obtained from Cayman chemicals, Michigan, USA. Wortmannin (PI3K inhibitor) and PD98059 (MEK1 inhibitor) were purchased from TOCRIS, R&D systems.
4
Inhibition of Mycobacterial-Induced Inflammation
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Heat-killed mycobacterium tuberculosis was purchased from InvivoGen (San Diego, CA, USA). The Met5A cells and mouse PMCs were treated with 10 ng/mL HKMT either with or without inhibitors for the time periods indicated. The PD98059 (MEK inhibitor), losartan (AT1R antagonist), and TAK-242 (TLR4 inhibitor) were purchased from R&D Systems (Minneapolis, MN, USA). To suppress endogenous NOX4 expression, a specific siRNA (BioneerInc, Daejeon, Korea) against NOX4 based on the target region from the NOX4 gene (sense: 5′-UCAGACAAAUGUAGACAC-3′ and antisense: 5′-AGUGUCUACAUUUGUCUG-3′) was used in experiments. Scrambled siRNA (sense: 5′-GGTCAAGACACTATTAACA-3′ and antisense: 5′-GGATTCCTAGTGTATTTCA-3′) was used as a control (SiCon).
5
Sphingosine-1-Phosphate Signaling Assays
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Sphingosine-1-Phosphate (d18:1; Lipid Maps LMSP01050001) was purchased from Avanti Polar Lipids and Sigma; bovine fatty acid free albumin was from Sigma; W146, CAY10444 and ML-031 were from Cayman Chemical; SEW2871 and CYM5541 were from Tocris Bioscience; LY294002, U0126 and PD98059 were from R&D systems.
6
Immune Signaling Modulation in PBMCs
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PBMCs from HIV-negative donors were stimulated with IL-12 and IL-15 in 96-well plates (24 h, 37 °C, 5% CO2). ERK inhibitor (PD98059, R&D), NFκB p65 inhibitor (PDTC, R&D), or NFAT inhibitor (480401-M, R&D) were added 6 h before harvesting. Cells were then harvested and analyzed by FlowJo™ 10.5.0 (URL: www.flowjo.com/flowjo-eula/ ).
7
Cannabinoid Signaling Pathway Analysis
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WIN 55,212-2, JWH-133, cannabidiol, MA and PD98059 were purchased from R&D Systems, Inc. (Wiesbaden-Nordenstadt, Germany). WIN 55,212-3 was from LGC Standards (Wesel, Germany). 2-AG was obtained from Cayman Chemical (Ann Arbor; Michigan, USA). AM-251, AM-630, AEA, arachidonic acid, SB203580 and SP600125 were bought from Enzo Life Sciences (Lörrach, Germany). Capsazepine, indometacin, myriocin from Mycelia sterilia (ISP-1), C2-ceramide (N-acetyl-D-sphingosine), dihydro-C2-ceramide (D-erythro-N-acetylsphinganine) and nSMase from Bacillus cereus were obtained from Sigma-Aldrich (Taufkirchen, Germany). IL-1β, penicillin-streptomycin and trypsin/EDTA were from Invitrogen Life Technologies (Darmstadt, Germany). THC and nSMase spiroepoxide inhibitor were from Lipomed (Weil am Rhein, Germany) and Santa Cruz Biotechnology (Heidelberg, Germany), respectively. Fumonisine B1 from Fusarium monoliforme was from Merck Millipore (Darmstadt, Germany). Fetal calf serum (FCS) was obtained from PAN Biotech GmbH (Aidenbach, Germany).
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