Nms 873

Manufactured by Selleck Chemicals

Sourced in United States

NMS-873 is a small molecule inhibitor that targets MDM2, a negative regulator of the tumor suppressor p53. It functions by binding to MDM2, preventing its interaction with p53 and thereby stabilizing and activating p53 signaling.

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Lab products found in correlation

Cb 5083, by Selleck Chemicals (4 mentions) Ml240, by Merck Group (3 mentions) Tunicamycin, by Merck Group (2 mentions) Dynasore, by Merck Group (2 mentions) Eeyarestatin 1, by Cayman Chemical (2 mentions) Dimethyl sulfoxide (dmso), by Merck Group (2 mentions) Chloroquine, by Merck Group (1 mentions) Hek293t cell, by American Type Culture Collection (1 mentions) Dmem medium, by Corning (1 mentions) Cycloheximide (chx), by Merck Group (1 mentions) Flavopiridol, by Merck Group (1 mentions) Foetal bovine serum (fbs), by Eurobio Scientific (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified, by Thermo Fisher Scientific (1 mentions) Calpain activity assay kit, by Abcam (1 mentions) Mln4924, by R&D Systems (1 mentions) Phenformin, by Merck Group (1 mentions) Selumetinib, by Selleck Chemicals (1 mentions) Calcium magnesium, by Thermo Fisher Scientific (1 mentions)

16 protocols using nms 873

Generation and Characterization of SEL1L CRISPR Null Cells

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HEK293T cells were purchased from ATCC. Hrd1 and SEL1L CRISPR null and control cells were described previously [28 (link)]. Cells were maintained in DMEM medium (Corning cellgro) containing 10% fetal bovine serum and penicillin–streptomycin. OS9, p97, SEL1L antibodies was purchased from Proteintech (10061-1-AP), Fitzgerald (10R-P104A), and Sigma (S3699), respectively. Cycloheximide and chloroquine were purchased from Sigma. MG132 was purchase from Calbiochem, NMS-873 was purchased from Selleckchem (S7285).
To generate SEL1L CRISPR null cells, the following primers corresponding to SEL1L 165-187 and 126-148 were synthesized and annealed. The double-stranded DNAs were cloned into pX330-U6-Chimeric_BB-CBh-hSpCas9 D10A vector [28 (link)].

SEL1L1 CR 1-F 5′-CACCGAGCTTGGCCTCGGCGTCCT

SEL1L1 CR 1-R 5′-AAACAGGACGCCGAGGCCAAGCTC

SEL1L1 CR 2-F 5′-CACCGCAGCAGCGTCAGCCCTATC

SEL1L1 CR 2-R 5′-AAACGATAGGGCTGACGCTGCTGC

Ye Y., Baek S.H., Ye Y, & Zhang T. (2018). Proteomic characterization of endogenous substrates of mammalian ubiquitin ligase Hrd1. Cell & Bioscience, 8, 46.

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Preparing Stock Solutions for Cell-Based Assays

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CB-5083 (Selleckchem, Houston, TX, USA, S8101), ML240 (Sigma-Aldrich, SML1071), DBeQ (Sigma-Aldrich, SML0031), NMS-873 (Selleckchem, S7285) and UPCDC30245 (Sigma-Aldrich, SML1674) were dissolved in DMSO to make a 50 mM stock solution. Tunicamycin (Sigma-Aldrich, T7765) was dissolved in DMSO to make 10 mM stock solution. The stock solution was aliquoted and stored at −80 °C, which was further diluted before making the final concentrations of the compound in culture media.

Bastola P., Wang F., Schaich M.A., Gan T., Freudenthal B.D., Chou T.F, & Chien J. (2017). Specific mutations in the D1–D2 linker region of VCP/p97 enhance ATPase activity and confer resistance to VCP inhibitors. Cell Death Discovery, 3, 17065-.

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HeLa, XP4PA-SV, and U2OS cell cultures

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HeLa (American Type Culture Collection ATCC CCL-2, human cervical carcinoma, female), XP4PA-SV (Coriell Institute for Medical Research, GM15983, human XPC-deficient skin fibroblasts, male), and U2OS cells (ATCC HTB-96, human osteosarcoma, female) were grown at 37 °C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% foetal bovine serum (EUROBIO), 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies). U2OS cells stably expressing H3.3-SNAP^{95 (link)} were maintained in the same medium in the presence of 100 μg/ml G418 (Invitrogen).
The VCP inhibitor NMS-873 (5 μM final concentration, Selleckchem) and the transcription inhibitor Flavopiridol (10 μM final concentration, Sigma-Aldrich) were added to the culture medium 2 h before UV irradiation of the cells and kept until the time of cell fixation.

Bouvier D., Ferrand J., Chevallier O., Paulsen M.T., Ljungman M, & Polo S.E. (2021). Dissecting regulatory pathways for transcription recovery following DNA damage reveals a non-canonical function of the histone chaperone HIRA. Nature Communications, 12, 3835.

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In Vitro TCF11/Nrf1 Digestion

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To digest TCF11/Nrf1 in vitro, the membrane fraction was isolated from cell pellets. First, pellets were resuspended in buffer 1 (50 mM Tris, pH 7.7; 50 mM NaCl; 5 mM MgCl₂) and the cells were lysed by three freeze–thaw cycles in liquid nitrogen. The cytosolic fraction was removed by centrifugation for 10 min at 1000×g, 4°C. The remaining pellet was resuspended in buffer 2 (buffer 1 + 1% NP-40) and incubated on ice for 30 min, followed by centrifugation for 10 min at 6000×g, 4°C. The supernatant contained the membrane fraction, which was used for the digestion.
For the digestion, the components of the Calpain Activity Assay Kit (ab65308, Abcam) were used. To the isolated membrane fraction, the Reaction Buffer and Active Calpain I as well as different inhibitors such as the impermeant Calpain Inhibitor Z-LLY-FML from the Calpain Activity Assay Kit, BTZ or NMS-873 (SelleckChem) were added. The mix was incubated for 30 min at 37°C before sample buffer was added to the sample and it was heated for 10 min to 95°C.
For the digestion using the proteasome, the membrane fraction was mixed with proteasome test buffer (50 µM Tris, 5 µM MgCl₂ and 2 µM ATP) and 0.5 µg proteasome isolated from human erythrocytes were added. The mix was also incubated for 30 min at 37°C before adding the sample buffer and heating the samples to 95°C for 5 min.

Nowak K., Taubert R.M., Haberecht S., Venz S, & Krüger E. (2018). Inhibition of calpain-1 stabilizes TCF11/Nrf1 but does not affect its activation in response to proteasome inhibition. Bioscience Reports, 38(5), BSR20180393.

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Inhibitor Screening for Deubiquitinases

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Inhibitors used in this work were DBeQ and NMS-873 (Selleckchem, Houston, TX), DUBs-IN-3/compound 22c (Medchemexpress, South Brunswick Township, NJ), and MLN4924 (Boston Biochem, Cambridge, MA).

McHugh A., Fernandes K., Chinner N., Ibrahim A.F., Garg A.K., Boag G., Hepburn L.A., Proby C.M., Leigh I.M, & Saville M.K. (2020). The Identification of Potential Therapeutic Targets for Cutaneous Squamous Cell Carcinoma. The Journal of Investigative Dermatology, 140(6), 1154-1165.e5.

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Radioiodide Uptake Assay Drugs

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Drugs used to treat cells in radioiodide uptake assays are listed in the key resources table. Chloroquine diphosphate was resuspended in PBS without calcium/magnesium (Thermo Fisher); Phenformin HCL in 100% ethanol and all other drugs in dimethyl sulfoxide (DMSO; Sigma-Aldrich), before being diluted in RPMI-1640 medium (Life Technologies) and added to cells at 1:100 dilution. Drugs reported to enhance NIS function and used as positive controls were DBeQ (Sigma-Aldrich), Dynasore (Sigma-Aldrich), Eeyarestatin-1 (Cayman Chemicals), NMS-873 (SelleckChem), Selumetinib (SelleckChem) and Vemurafenib (SelleckChem).

Read M.L., Brookes K., Thornton C.E., Fletcher A., Nieto H.R., Alshahrani M., Khan R., Borges de Souza P., Zha L., Webster J.R., Alderwick L.J., Campbell M.J., Boelaert K., Smith V.E, & McCabe C.J. (2022). Targeting non-canonical pathways as a strategy to modulate the sodium iodide symporter. Cell Chemical Biology, 29(3), 502-516.e7.

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Inhibition of VCP and Dynamin in Cells

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All drugs were resuspended in dimethyl sulfoxide (DMSO), diluted in RPMI, and then added directly to cells at the appropriate final concentration. Cells were treated with VCP inhibitors: Eeyarestatin-1 (ES-1; Cayman Chemicals), NMS-873 (SelleckChem), astemizole (Sigma-Aldrich), clotrimazole (Sigma-Aldrich), and ebastine (Sigma-Aldrich) or the dynamin inhibitor dynasore (Sigma-Aldrich).

Fletcher A., Read M.L., Thornton C.E., Larner D.P., Poole V.L., Brookes K., Nieto H.R., Alshahrani M., Thompson R.J., Lavery G.G., Landa I., Fagin J.A., Campbell M.J., Boelaert K., Turnell A.S., Smith V.E, & McCabe C.J. (2019). Targeting Novel Sodium Iodide Symporter Interactors ADP-Ribosylation Factor 4 and Valosin-Containing Protein Enhances Radioiodine Uptake. Cancer research, 80(1), 102-115.

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Drosophila and Human Cell Drug Treatments

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For drug treatment of Drosophila, flies were collected after eclosion and divided into separate vials (20~25 flies each vial). Instant fly food (Carolina) was mixed with 1% H2O2 (cat#: H1009, Sigma-Aldrich), 250 μM rotenone (cat#: R8875, Sigma-Aldrich) or 100 μM CCCP (cat#: C2759, Sigma-Aldrich). Vials were changed every day. Samples were collected for further analyses after 5 days of treatment. In order to achieve high efficiency, anisomycin stock (100mM in DMSO, cat#: A9789, Sigma-Aldrich) were diluted with the Schneider’s Medium (cat#: 21720–024, GIBCO™) to 250 μM and injected into fly thorax between mesonotum and scutellum via a hand-made glass needle. After injection, flies were kept in the vial with standard fly food and waited for 24 hours (short-term) before the experiments, and for long-term (7 days) treatment, three injections (day0, day3, day6) were applied to newly eclosed flies.
For drug treatment in human cells, HeLa cells or HeLa/GFP-Parkin cells were cultured and treated with CCCP at the indicated concentrations and durations as described (Narendra et al., 2010 (link)). For most experiments, 20 μM CCCP concentration was used. For Antimycin/Oligomycin treatment, 10 μM of each compound was used. For anisomycin, 200 μM was used, and for the VCP inhibitor NMS-873 (cat#: S7285, Selleckchem), 1 μM was used.

Wu Z., Tantray I., Lim J., Chen S., Li Y., Davis Z., Sitron C., Dong J., Gispert S., Auburger G., Brandman O., Bi X., Snyder M, & Lu B. (2019). MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure. Molecular cell, 75(4), 835-848.e8.

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Organelle Protein Regulation Assay

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MG132 (Enzo Biochem), NMS873 (Selleck Chemicals), bortezomib, bafilomycin A1, and MLN4924 (EMD Millipore) were dissolved in DMSO, stored at −80°C, and used at the indicated concentrations. DMSO at 0.1% served as the vehicle control.
Antibodies used in this study were SLC25A46 (Proteintech); HA (HA.11; Biolegend); MFN1 (ab57602) and histidine (HIS.H8; Abcam); LC3A/B (Cell Signaling); MFN2 (D1E9; Cell Signaling); MTCH2 (Abgent); MIC60/IMMT (Abgent and Proteintech); MID51 (Proteintech); Ubiquitin (FK2, Enzo Biochem); Mortalin (University of California, Davis/National Institutes of Health NeuroMab Facility); MIC19/CHCHD3 (Everest Biotech); MIC27/Apool (Assay BioTech); OPA1, P97/VCP, and TIMM23 (BD Biosciences); TOMM20 (FL-145), LRP130 (H-300), and glyceraldehyde-3-phosphate dehydrogenase (FL-335; Santa Cruz Biotechnology); MULAN (HPA017681) and Flag (M2) (Sigma-Aldrich); MARCH5 (provided by Richard Youle); myc (9E10; Developmental Studies Hybridoma Bank, University of Iowa); and Cullin 4a (PA5-29857; Thermo Fisher Scientific). PREP, GFP, TOMM40, YME1L, and PNPase polyclonal antibodies were raised against recombinant proteins (Pacific Immunology).

Steffen J., Vashisht A.A., Wan J., Jen J.C., Claypool S.M., Wohlschlegel J.A, & Koehler C.M. (2017). Rapid degradation of mutant SLC25A46 by the ubiquitin-proteasome system results in MFN1/2-mediated hyperfusion of mitochondria. Molecular Biology of the Cell, 28(5), 600-612.

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Preparation of Stock Solutions for Cell Studies

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CB-5083 (S8101, Selleckchem, Houston, TX, USA), NMS-873 (S7285, Selleckchem), DBeQ (SML0031, Sigma-Aldrich, Saint Louis, MO, USA), STF-083010 (S7771, Selleckchem,), STF-083010 (SML0409, Sigma-Aldrich), ISRIB (SML0843, Sigma-Aldrich) and mifepristone (M8046, Sigma-Aldrich) were dissolved in dimethyl sulfoxide (DMSO) at 50 mM stock solution. Tunicamycin (T7765, Sigma-Aldrich) was dissolved in DMSO at 10 mM stock solution and ISRIB (SML0843, Sigma-Aldrich) was dissolved in DMSO at 5 mM stock solution. All stock solutions were aliquoted in small volumes and stored at −80 °C. Before use, appropriate concentrations of these compounds were prepared by dissolving the stock solution (or its subsequent dilution) into the appropriate growth media.

Bastola P., Leiserowitz G.S, & Chien J. (2022). Multiple Components of Protein Homeostasis Pathway Can Be Targeted to Produce Drug Synergies with VCP Inhibitors in Ovarian Cancer. Cancers, 14(12), 2949.

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