Candidate inhibitor compounds DBeQ, ML240, and NMS-873 were purchased from Sigma-Aldrich (St Louis, MO) and used directly without further purification; C3 and C6 were obtained from TimTec (Newark, DE). The compounds in a powder form were dissolved in DMSO at 10 mM and used in biochemical studies after further dilutions.
Nms 873
Manufactured by Merck Group
Sourced in United States
NMS-873 is a laboratory equipment product manufactured by Merck Group. It is designed for use in scientific research and analysis. The core function of NMS-873 is to perform a specific set of tasks within a controlled laboratory environment, facilitating data collection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Merck Group (3 mentions)
S trityl l cysteine, by Merck Group (2 mentions)
R mg132, by Cayman Chemical (2 mentions)
Rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Carfilzomib, by Selleck Chemicals (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ml240, by Merck Group (1 mentions)
Mcm7 sc 9966, by Santa Cruz Biotechnology (1 mentions)
Laminb1 ab16048, by Abcam (1 mentions)
Pd166285, by Merck Group (1 mentions)
Phospho chk1 ser345, by Cell Signaling Technology (1 mentions)
Vx 445, by Selleck Chemicals (1 mentions)
Tak 243, by MedChemExpress (1 mentions)
Vx 661, by Selleck Chemicals (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
Sumo 1, by Santa Cruz Biotechnology (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Hek293, by Thermo Fisher Scientific (1 mentions)
14 protocols using nms 873
1
Purification and Use of Inhibitor Compounds
2
Cell Cycle Regulation Antibodies and Inhibitors
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Antibodies to Psf2, XCAP-E, claspin, and Cut5/TopBP1 were prepared as per directions (23 (link), 52 (link), 53 (link)). Antisera to Cdc45 and Polε (p60) were provided by H. Takisawa and Y. Kubota (Osaka University). Antiserum to Polδ (p66) was provided by S. Waga (Japan Women’s University). Antibodies to APC3 and Cyclin B2 were provided by S. Mochida (Kumamoto University). The following antibodies were obtained from the indicated companies: MCM7 (sc-9966, Santa Cruz), MCM4 (ab4459, Abcam), lamin B1 (ab16048, Abcam), β-actin (ab8224, Abcam), Phospho-Chk1 (Ser345) (2341, Cell Signaling), Phospho-CDK1 (Thr14, Tyr15) (44-686G, Thermo Fisher). Recombinant His-p27 and His-geminin were prepared as described (50 (link)) and used at 100 μg/ml and 50 μg/ml to inhibit CDK activities and replication licensing, respectively. NMS-873 (Sigma-Aldrich) and PD166285 (Sigma-Aldrich) were used at 100 μM and 10 μM to inhibit p97/VCP and Wee1/Myt1 kinase activities, respectively.
3
Monitoring CMG Ubiquitylation and Unloading
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Xenopus egg extracts were prepared as described (18 (link)). Mock and CRL2Lrr1 immunodepletions were performed as previously described (7 (link),8 (link)). To monitor CMG ubiquitylation and unloading, as well as CRL2Lrr1 recruitment (Figure4A ), 45 ng of plasmid DNA was incubated in a high-speed supernatant of egg cytoplasm followed by addition of nucleoplasmic extract (NPE) in the presence or absence of 200 μM p97 inhibitor (p97i; NMS-873, Sigma #SML1128). The extracts were mock depleted, Lrr1 depleted or Lrr1 depleted and supplemented with ∼45 nM rCRL2Lrr1 or rCRL2Lrr1ΔPH. After 45 min, plasmids were pulled down as described previously, and the chromatin was subjected to western blotting (19 (link)). To assess CMG ubiquitylation in extracts lacking added DNA (Figure 4B ), we monitored Mcm7 ubiquitylation following the addition of 15 nM rCMG into NPE in the presence of 45 μM His-ubiquitin and 180 μM p97i. Extracts were mock depleted, Lrr1 depleted or Lrr1 depleted and rescued with ∼65 nM rCRL2Lrr1 or rCRL2Lrr1ΔPH. After 40 min, His-ubiquitin was pulled down as previously described and the recovered material subjected to western blotting (7 (link)). Experiments were performed at least three times. A representative example is shown.
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Xenopus egg extracts were prepared as described (18 (link)). Mock and CRL2Lrr1 immunodepletions were performed as previously described (7 (link),8 (link)). To monitor CMG ubiquitylation and unloading, as well as CRL2Lrr1 recruitment (Figure
4
Inhibitor Kinetics and Corrector Assays
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For degradation kinetics and steady-state experiments with inhibitors, reporter cells were induced with 1 μg/ml dox for 16 h then dosed with 20 μM NMS-873 (Sigma-Alrich), 2 μM TAK-243 (Med Chem Express, Monmouth Junction, NJ), or 1 μM BTZ, (Selleck Chemicals, Houston, TX) for 3 h. For experiments with corrector drugs tezacaftor (VX-661, 5 μM, Selleck Chemicals), and/or elexacaftor (VX-445, 5 μM, Selleck Chemicals) were coadministered with 1 μg/ml dox for 16 h.
5
Cell Culture Conditions for Glioma and HEK293 Cells
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HEK293 and HEK293T cell lines were purchased from American Type Culture Collection. Several glioma stem-like cell lines derived from both primary (GSC923) and recurrent (GSC827, GSC604) glioblastoma tumors and normal murine neural stem cells (MNSC) were described previously31 (link). All cells were grown at 37 °C in 5% CO2. HEK293 cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine and 1% penicillin-streptomycin (Gibco), and the GSC and MNSC were cultured in NBE complete media31 (link). Chemical such as MG132, N-ethylmaleimide, NMS-873, cyclohexamide and ATRA were purchased from Sigma-Aldrich. Puromycin was from Life Sciences. Antibodies used in Western blots and immunoprecipitations included: RARA, RXRA, Sumo1, Sumo2, Ub, VCP from Santa Cruz Biotechnology, DDK from Origene, β-actin from Sigma-Aldrich, normal rabbit IgG and normal mouse IgG from Dako, and Histone H3 from Cell Signaling.
6
Proteasomal Inhibition and Stress Response
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7
Xenopus Egg Extract DNA Replication
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Animal work performed at Harvard Medical School was approved by the Harvard Medical Area Standing Committee on Animals (HMA IACUC Study ID IS00000051-6, approved 10/23/2020). The institution has
an approved Animal Welfare Assurance (D16-00270) from the NIH Office of Laboratory Animal Welfare. Xenopus egg extracts were prepared as described in the protocol68 . For DNA replication, plasmids were licensed by incubation in high-speed supernatant of egg cytoplasm for 30 min at room temperature at a final concentration of 15 ng pKV45/µl extract (plasmid-pulldown assays) or 7.5 ng pBluescript II/ µl extract (chromatin immunoprecipitation assay). Replication was initiated by addition of two volumes of nucleoplasmic egg extract to licensing mixture. In all figures, the addition of nucleoplasmic egg extracts corresponds to the zero-minute time point. Where indicated, nucleoplasmic egg extracts were supplemented with p97i, NMS-873 (Sigma) or Culi, MLN4924 (Active Biochem), to a final concentration of 200 µM each and incubated for 5 min prior to initiation of DNA replication, unless otherwise indicated. Reactions were supplemented with approximately 140 nM recombinant FLAG-Ubxn7, 50–140 nM of recombinant FLAG-Faf1, and 80 nM recombinant Ufd1-Npl4.
an approved Animal Welfare Assurance (D16-00270) from the NIH Office of Laboratory Animal Welfare. Xenopus egg extracts were prepared as described in the protocol68 . For DNA replication, plasmids were licensed by incubation in high-speed supernatant of egg cytoplasm for 30 min at room temperature at a final concentration of 15 ng pKV45/µl extract (plasmid-pulldown assays) or 7.5 ng pBluescript II/ µl extract (chromatin immunoprecipitation assay). Replication was initiated by addition of two volumes of nucleoplasmic egg extract to licensing mixture. In all figures, the addition of nucleoplasmic egg extracts corresponds to the zero-minute time point. Where indicated, nucleoplasmic egg extracts were supplemented with p97i, NMS-873 (Sigma) or Culi, MLN4924 (Active Biochem), to a final concentration of 200 µM each and incubated for 5 min prior to initiation of DNA replication, unless otherwise indicated. Reactions were supplemented with approximately 140 nM recombinant FLAG-Ubxn7, 50–140 nM of recombinant FLAG-Faf1, and 80 nM recombinant Ufd1-Npl4.
8
Preparation of Inhibitor Stock Solutions
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Stock solutions were prepared for the following inhibitors: bortezomib (LC Laboratories; catalog no.: B-1408; 100 mM in dimethyl sulfoxide [DMSO]), NMS-873 (Sigma; catalog no.: SML1128; 10 mM in DMSO), pifithrin μ (Selleckchem; catalog no.: S2930; 20 mM in DMSO), TAK-243 (Active Biochem; catalog no.: A1384; 10 mM in DMSO), nelfinavir (Cayman Chemical; catalog no.: 15144; 20 mM in methanol), and Ub-aldehyde (Boston Biochem; catalog no.: U201; 1600 μM in 50 mM HCl).
9
Xenopus Cell Cycle Regulation
10
Synchronizing and Enriching Mitotic Cells
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HeLa cells were first synchronized in S phase by addition of thymidine (at a final concentration of 2 mM) for 24 h. S phase cells were washed with 1×PBS to remove excess thymidine and released into fresh media (DMEM/10% FBS) for 3 h. To arrest cells in prometaphase, released cells were treated with S-trityl-L-cysteine (Sigma, 164739) (at a final concentration of 5 μM) for 12–14 h. Finally, prometaphase cells were collected by vigorous pipetting, washed with 1×PBS, and used for downstream applications, including immunoprecipitation assays and/or Western blot analyses, or frozen in liquid nitrogen and stored at −80°C for later use. For cell cycle studies, prometaphase cells were released into fresh media and collected at indicated time points. For drug inhibition studies, cells were released into media containing 2 μM carfilzomib (Selleck, PR-171), 20 μM (R)-MG132 (Cayman, 13697) and/or 10 μM NMS-873 (Sigma, SML1128) for indicated times. For depletion studies, HeLa cells were transfected with 40 nM of indicated siRNAs and a 1:400 dilution of RNAiMAX transfection reagent (Thermo Fisher, 13778150) 24 h prior to synchronization.
Mitotic enrichment of HEK 293Ts and H1s was achieved by adding STLC (at a final concentration of 5 μM) to the culture media for 14–16 h.
Mitotic enrichment of HEK 293Ts and H1s was achieved by adding STLC (at a final concentration of 5 μM) to the culture media for 14–16 h.
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