BMMCs (106 cells/ ml) were seeded in IL‐3‐free media and were subsequently treated with inhibitors SP600125 (1 µM (0.01% DMSO), Merck), SB203580 (1 µM (0.01% DMSO), Merck), UO126 (5 µM (0.1% DMSO), Merck), IKK‐16/IKKiVII (1 µM (0.1% DMSO), Merck), PF3644022 (10 µM (0.1% DMSO), Merck) or its representative DMSO concentration as a control. Upon 30 min of incubation, all further stimulations were added to the culture as indicated.
Pf 3644022
Manufactured by Merck Group
Sourced in United Kingdom, United States
PF-3644022 is a laboratory equipment product offered by Merck Group. It is a precision instrument designed for specific laboratory applications. The core function of this product is to facilitate various experimental and analytical procedures within a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Anisomycin, by Merck Group (3 mentions)
Cycloheximide (chx), by Merck Group (3 mentions)
Sp600125, by Merck Group (2 mentions)
Ph 797804, by Selleck Chemicals (2 mentions)
Berzosertib, by Selleck Chemicals (2 mentions)
Doxycycline, by Thermo Fisher Scientific (2 mentions)
Jnk in 8, by Selleck Chemicals (2 mentions)
Sb202190, by Merck Group (2 mentions)
Bestatin methyl ester, by Abcam (2 mentions)
Lactimidomycin, by Merck Group (2 mentions)
Doxorubicin, by Selleck Chemicals (2 mentions)
Ku 60019, by Selleck Chemicals (2 mentions)
Vx 765, by Selleck Chemicals (2 mentions)
Mln7243, by Chemietek (2 mentions)
Doramapimod, by Cayman Chemical (2 mentions)
Mln4924, by MedChemExpress (2 mentions)
Isrib, by Selleck Chemicals (2 mentions)
Bafilomycin a1, by Merck Group (2 mentions)
Bortezomib, by Selleck Chemicals (2 mentions)
12 protocols using pf 3644022
1
Inhibitor Effects on BMMCs
2
Kinase Inhibitors in Biological Assays
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The protein kinase inhibitor SB203580 was obtained from Tocris Chemical Co. (Bristol, UK). The kinase inhibitors BIRB 796 and UR-13756 were synthesised according to (Bagley et al. 2006 (link), 2010 (link)). The MK2 inhibitors MK2.III (Anderson et al. 2007 (link)) and PF-3644022 (Mourey et al. 2010 (link)) were obtained from Merck (UK) and Tocris Chemical Co. (Bristol, UK) respectively.
3
Cellular Signaling Pathway Modulation
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Penicillin/streptomycin (#P0781), DMSO (#472301), anisomycin (# A5862), LPS, Sodium arsenite, PF-3644022 (#PZ0188) were purchased from Sigma-Aldrich, MO, USA. SB-202190 (#BML-EI294-001), PD-184352 (#ALX-270-471) were purchased from Alexis Biochemicals, CA, USA. Recombinant human TNF-α (#300-01A) was purchased from PeproTech, NJ, USA. Lambda phosphatase was purchased from New England Biolabs, MA, USA, BI-605906 was purchased from R&D systems, UK.
4
Diverse Cell Line Culture and Treatments
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U2OS, A549, Saos-2, and HEK293 cells (purchased from ATCC) were cultured either in Dulbecco’s modified Eagle medium (Sigma, D5796) or Roswell Park Memorial Institute Medium (RPMI)-1640 supplemented with 10% fetal bovine serum (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 PH797804 (Selleckchem, S2726) or 500 nm BIRB0796 (Axon MedChem, 1358), and 200 nm LY2228820 (Selleckchem, S1494). MK2 was inhibited using 10 μm MK2 Inhibitor III (Calbiochem, 475864–5MG) or 10 μm PF 3644022 (Sigma, PZ0188). The following additional treatments were used: 0.5 μm MitoQ (kindly provided by M. Murphy, Cambridge, UK), 250 nm Doxorubicin hydrochloride (Sigma, D1515–10mg), 50 μm Z-VAD (OMe)-FMK (SM Biochemicals LLC SMFMK001), 200 or 50 nm bafilomycin A1 (Santa Cruz, sc-201550), 20 μm chloroquine (Sigma, C6628), 10 μm Spautin-1 (Axon MedChem, 2512), 15 μm palbociclib (Selleckchem, S1116), 500 nm camptothecin (Sigma, C9911). The compounds were dissolved in dimethyl sulfoxide (DMSO) or water and the total concentration of DMSO in the culture medium never exceeded 1%.
5
TNF-Induced Septic Shock in Mice
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Wild‐type or Pml‐knockout C57BL/6 mice aged 6–8 weeks and of the same sex were used for TNF‐induced septic shock. Mice were anesthetized using Avertin, and mouse TNF (1.5 µg/g) in a total volume of 200 µl endotoxin‐free PBS was injected intravenously (i.v.). Body temperatures were monitored rectally every 1–3 h for 30 h using an industrial electronic thermometer (Kane‐May), and mouse mortality was recorded at the same time. Mice were sacrificed when their body temperature fell below 22°C. Serum was collected by cardiac puncture 15 min within mice dying. For live mice, serum was collected 30 h after TNF injection. Serum LDH was measured using an LDH Cytotoxicity Detection Kit (Clontech, #MK401) following the user manual. Serum GPT/ALT levels were determined using FUJI DRI‐CHEM SLIDE GPT/ALT‐P III (FUJIFILM, Tokyo, Japan). Mouse serum TNF, IL‐6, and IL‐1α levels were detected using a TNF alpha Mouse Uncoated ELISA Kit (Invitrogen, #88‐7324‐88), IL‐6 Mouse Uncoated ELISA Kit (Invitrogen, #88‐7064‐88), or ELISA MAX™ Deluxe Set Mouse IL‐1α (BioLegend, #433404), respectively. For MK2 and RIPK1 inhibitor treatment, mice were pre‐treated with 75 µg PF‐3644022 (Sigma‐Aldrich, # PZ0188) per mouse or Nec‐1s (6 µg/g) 15 min before TNF injection by intraperitoneal injection, and then subjected to inhibitor treatment again 60 min after TNF injection.
6
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%.
7
Immune Cell Characterization Protocol
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DNFB, brefeldin A, and PF3644022 were from Sigma-Aldrich; SC409, PD98059, SP600125, and rapamycin from EMD Millipore; CFSE from Life Technologies; IFN-γ, IL-2, IL-4, IL-6, and TGF-β from Peprotech. Fluorescent-conjugated antibodies against the following markers were used in flow cytometry: CD3 (145–2C11), CD4 (GK1.5 and RM4–5), CD8 (53–6.7), CD25 (PC61.5), Foxp3 (anti-mouse/rat staining kit), IFN-γ (XMG1.2), IL-4 (BVD6–24G2), IL-17A (eBio17B7; all from eBioscience). Antibodies against the following proteins were used in cell stimulation, Fc receptor blocking, and cytokine neutralization: CD3 (145–2C11), CD28 (37.51), IFN-γ (XMG1.2), IL-12 (C17.8), IL-4 (11B11), CD16/CD32 (2.4G2; all from BD Pharmingen); and hamster IgG (MP Biomedicals). Antibodies against the following proteins were used in immunoblotting: ERK (9102), phosphorylated (p-) p38 (9211), p-JNK (9251), p-ERK (9101), p-S6K1 (9205), p-S6 (4858), p-MK2 (3007), p-TSC2 (3616; all from Cell Signaling Technology); p38α (sc-535; Santa Cruz Biotechnology); p38β (33–8700; Life Technologies); p38γ and p38δ (University of Dundee); JNK (554285, BD Pharmingen); Foxp3 (14–5773, eBioscience); and actin (A4700; Sigma-Aldrich).
8
Cytokine Profiling of Intestinal Tissues
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CD and UC tissues were collected under IRB-approved protocols at the University of New Mexico (10-513) for discarded surgical resections and 00127500 for the use of biopsy samples from the GI and IBD Tissue Bank at the University of Utah. Samples were collected from patients with no GI pathologies or CD with active disease or in remission. Sample data are provided in a de-identified manner in Table 1 . Tissue samples were divided into 4 mg +/− 0.3 mg and incubated in RPMI complete media for 18 h. Some samples were incubated with MK2 inhibitor (PF-3644022, Sigma Aldrich) at 50 μM, the MMP inhibitor GM6001 at 200 μM, or vehicle (DMSO) control for 18 h, and supernatants were collected for multiplex analysis.
9
Small Molecule Inhibitors for Cellular Studies
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The following small compound inhibitors and reagents were used: 2-Deoxy-D-glucose (Sigma-Aldrich), anisomycin (Sigma-Aldrich), bafilomycin A1 (Sigma-Aldrich), berzosertib (Selleckchem), bestatin methyl ester (Abcam), blasticidin (InvivoGen), bortezomib (Selleckchem), cycloheximide (Sigma-Aldrich), deoxyribonucleic acid sodium salt from herring testes (Sigma-Aldrich), doramapimod (Cayman), doxycycline (Thermo Fisher Scientific), doxorubicin (Selleckchem), etoposide (Calbiochem), H2O2 (Roth), ISRIB (Selleckchem), JNK-IN-8 (Selleckchem), KU-60019 (Selleckchem), lactimidomycin (Sigma-Aldrich), LPS-EK Ultrapure (Invivogen), MG-132 (Selleckchem), MLN4924 (MedChem Express), MLN7243 (ChemieTek), Nigericin sodium salt (Biomol), PF-3644022 (Sigma-Aldrich), SB202190 (Sigma-Aldrich), poly(dA:dT) (Invivogen), poly(I:C) low molecular weight and high molecular weight (Invivogen), sodium azide (Sigma-Aldrich), talabostat mesylate (MedChemExpress), Vx-765 (Selleckchem), and Z-VAD(Ome)-FMK (MedChemExpress). ZAKα inhibitor 6p (Yang et al., 2020 (link)) was kindly provided by IFM Therapeutics. 6p was synthesized and characterized by 1H NMR spectroscopy and LC-MS, with the data being fully consistent with literature values for this compound.
10
UV-Induced Stress Response Pathways
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Cells were pretreated with 10 µM of p38 inhibitor SB203580 (Selleckchem) or MK2/3/5 inhibitor PF-3644022 (Sigma-Aldrich) for 1 h before irradiation with UV light (40 J/m2). After 1 h recovery period, cells were washed with ice-cold phosphate-buffered saline (PBS). Cells were lysed in modified RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% sodium deoxycholate) supplemented with protease inhibitors (Complete protease inhibitor cocktail tablets, Roche Diagnostics), 1 mM sodium orthovanadate, 5 mM β-glycerophosphate, and 5 mM sodium flouride (all from Sigma). Subsequently, lysates were cleared by centrifugation at 16,000 × g for 15 min and protein concentrations were estimated using QuickStart Bradford Protein assay (BioRad).
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