To determine the pathways involved in IL-17 induced B cell migration, we used pharmacologic inhibitors directed against various pathways that are potentially known to be activated by IL-17 and may be involved in cells migration. B cells (50×103) were incubated with the inhibitors for 1 hour at 37°C prior to migration assay. Specifically, we used the p38 MAPK inhibitor SB203580 (0.1 mM; Axon Medchem, Groningen, The Netherlands), the extracellular signal-regulated kinase (ERK) 1/2 MAPK inhibitor PD184352 (2 mM; United States Biological, Inc, Swampscott, Mass), the NF-kB inhibitor PS1145 (10 mM; Professor Sir Philip Cohen), and the phosphoinositide 3-kinase (PI3K) inhibitor PI103 (5 mM; Cayman Chemical, Ann Arbor, Mich). All results were compared with the corresponding vehicle control dimethyl sulfoxide.
Sb203580
Manufactured by Axon Medchem
SB203580 is a selective inhibitor of p38 mitogen-activated protein kinase (p38 MAPK). It functions by blocking the enzymatic activity of p38 MAPK, a key signaling molecule involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pi 103, by Cayman Chemical (1 mentions)
Pd184352, by US Biological (1 mentions)
Ps 1145, by Cayman Chemical (1 mentions)
Gsk j4, by Bio-Techne (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Anisomycin, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Mg132, by Peptide Institute (1 mentions)
Penicillin g, by Thermo Fisher Scientific (1 mentions)
Escherichia coli 0127 b8, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Birb796, by Axon Medchem (1 mentions)
Cgp57380, by Merck Group (1 mentions)
Pf 3644022, by Merck Group (1 mentions)
Ph 797804, by Selleck Chemicals (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Ph 797804, by Axon Medchem (1 mentions)
4 protocols using sb203580
1
Investigating IL-17 Induced B Cell Migration
2
Hippocampal Neuron NMDA-LTD Inhibition
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Stock solutions of CBP-CREB Interaction Inhibitor (250 mM (CCIIh); Millipore ref. 217505), KDM6A/B inhibitor (GSK-J4; 100 mM; Tocris Bioscience ref. 4594) and p-38 MAK inhibitor (SB203580; 100 mM; Axon Medchem ref. Axon 1363) were prepared in dimethyl sulfoxide (Sigma). NMDA (20 mM; Sigma-Aldrich ref. M3262) was prepared in Milli-Q water. Hippocampal neurons were treated with 5 μM CCIIh (1 h), 10 μM GSK-J4 (1 h) or 10μM SB203580 (1 h) before NMDA-LTD induction. LTD was induced by 20 μM NMDA for 5 min, and the medium was replaced. Samples were collected 5 or 25 min after LTD induction. Samples without LTD induction were used as a control.
3
Cell Culture and Inhibitor Treatments
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Cells were grown in DMEM (HeLa and HEK293T) on plates or in RPMI1640 (THP-1) as a suspension containing 2 mM l -glutamine, 10% (v/v) FCS, 100 units penicillin G/mL, and 100 mg/mL streptomycin (all purchased from Life Technologies) with 5% CO2 in a humidified atmosphere at 37°C. The HeLa cells were kindly provided by H. Holtmann (Hannover, DE). All cells were treated as indicated with 10 µg/mL Anisomycin (dissolved in DMSO, Sigma), 1 µg/mL lipopolysaccharide (LPS, dissolved in water, Escherichia coli 0127:B8, Sigma), or pretreated for a minimum of 45 min with BIRB796, SB203580, and SB202190 (all dissolved in DMSO, Axon Medchem) at concentrations of 1 µM (BIRB796) and 5 µM (SB compounds), respectively. For inhibition of the proteasome, cells were incubated with 20 µM MG-132 (dissolved in DMSO, Peptide Institute). The MK2-inhibitor PHA78108 was a kind gift from Robert J. Mourey (Pfizer).
4
Pharmacological Inhibition of p38 Signaling
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U2OS cells (purchased from ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM, Sigma, D5796) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific, E6541L), 2 mM L-Glutamine (LabClinics, M11–004) and 100 μg/ml penicillin-streptomycin (LabClinics, P11-010). For the inhibition of p38α, we used 1 μM PH-797804 (Selleckchem, S2726) or 10 μM SB203580 (Axon MedChem); both compounds can also inhibit p38β, although PH-797804 has been reported to preferentially inhibit p38α44 (link). MK2 was inhibited using 10 μM MK-2 Inhibitor III (Calbiochem) or 10 μM PF 3644022 (Sigma, PZ0188), whereas MSK activity was inhibited using 5 μM SB 747651 (Axon MedChem), and MNK activity was inhibited using 10 μM CGP-57380 (Sigma). MitoQ (kind gift from M. Murphy, Cambridge, UK) was used at 0.5 μM. Cisplatin was used at 50 μM, and the caspase inhibitors Z-VAD(OMe)-FMK (SM Biochemicals LLC SMFMK001) and Q-VD-OPH (SM Biochemicals LLC SMPH001) at 50 μM. The inhibitors were dissolved in DMSO and the total concentration of DMSO in the culture medium did not exceed 1%.
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