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Mln4924

Manufactured by Selleck Chemicals
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MLN4924 is a small molecule that inhibits the NEDD8-activating enzyme (NAE). NAE is an essential component of the neddylation pathway, which is involved in the regulation of various cellular processes. MLN4924 functions by binding to and inactivating NAE, thereby disrupting the neddylation pathway and leading to the accumulation of neddylated proteins.

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Lab products found in correlation

Mg132, by Selleck Chemicals (13 mentions) Cycloheximide (chx), by Merck Group (12 mentions) Bortezomib, by Selleck Chemicals (9 mentions) Dimethyl sulfoxide (dmso), by Merck Group (5 mentions) Mg132, by Merck Group (4 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Cycloheximide (chx), by Selleck Chemicals (3 mentions) Anti flag, by Merck Group (3 mentions) Phospho s6k t389, by Cell Signaling Technology (2 mentions) Phospho akt t308, by Cell Signaling Technology (2 mentions) Flag tag (flag), by Merck Group (2 mentions) Fdft1, by Abcam (2 mentions) β tubulin, by Merck Group (2 mentions) Phospho akt s473, by Cell Signaling Technology (2 mentions) Src 3, by BD (2 mentions) Dhcr24, by Abcam (2 mentions) Dhcr7, by Abcam (2 mentions) Igf1r, by Santa Cruz Biotechnology (2 mentions) I bet762 i bet, by MedChemExpress (2 mentions) Mk 2206, by Selleck Chemicals (2 mentions)

Spelling variants of this product

MLN4924 (S7109) (2 citations)

52 protocols using mln4924

1

Spinal Cord Ischemia-Reperfusion Injury Model

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For the in vivo experiments, the animals were assigned a unique number and then randomly divided into groups (n = 6 per group). The sham group underwent the surgical procedure without aortic clipping. The I/R group received abdominal aortic exposure and cross-clamping for 60 min followed by intraperitoneal injection of an equivalent volume of 0.9% saline solution immediately after reperfusion. The rats in the I/R + MLN4924 (S7109, Selleck Chemicals) (n = 6) and I/R + MLN4924 + EX527 (S1541, Selleck Chemicals) (n = 6) groups also received the same surgical procedure as those in the I/R group but were treated with MLN4924 immediately after I/R injury. EX527 was injected 0.5 h before the onset of SCIR in the I/R + MLN4924 + EX527 group. All experiments were repeated three times.
Yu S., Xie L., Liu Z., Li C, & Liang Y. (2019). MLN4924 Exerts a Neuroprotective Effect against Oxidative Stress via Sirt1 in Spinal Cord Ischemia-Reperfusion Injury. Oxidative Medicine and Cellular Longevity, 2019, 7283639.
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2

Preparing Inhibitor MLN4924 Solution

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We dissolved MLN4924 (1 mM, in DMSO) (Selleckchem., Houston, TX, USA) to medium or egg water and yielded a final MLN4924 concentration of 1 μM. Controls were treated equivalent volumes of DMSO.
Yu G., Liu X., Tang J., Xu C., Ouyang G, & Xiao W. (2019). Neddylation Facilitates the Antiviral Response in Zebrafish. Frontiers in Immunology, 10, 1432.
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3

Immunoprecipitation of Ubiquitin-Conjugated Proteins

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Following treatment with DMSO, 1 μM MLN4924 (Selleck Chemicals), or 100 nM MLN7243 for 24 hours, cells were harvested by trypsinization, washed with PBS, and lysed in buffer DLB (50 mM Tris-Cl and 2% SDS with 5 mM DTT and protease inhibitors added fresh) with rotation for 20 minutes at 4°C. DNA was sheared by sonication to reduce viscosity. Samples were heated at 95°C for 10 minutes. Lysates were cleared by centrifugation, and then diluted 20-fold in DNB (50 mM Tris-Cl, 150 mM NaCl, 5 mM EDTA, 1% NP-40, and 0.5% sodium dodecyl sulfate with fresh protease inhibitors). Lysates were pre-cleared with protein A/G beads (Santa Cruz), and then incubated overnight with desired antibodies or corresponding IgG controls at 4°C with rotation. The following day lysates were incubated with protein A/G beads 4°C with rotation for 2 hours, washed extensively in DNB, and eluted by boiling in 2x Laemmli sample buffer.
McGrail D.J., Garnett J., Yin J., Dai H., Shih D.J., Lam T.N., Li Y., Sun C., Li Y., Schmandt R., Wu J.Y., Hu L., Liang Y., Peng G., Jonasch E., Menter D., Yates M.S., Kopetz S., Lu K., Broaddus R., Mills G.B., Sahni N, & Lin S.Y. (2020). Proteome instability is a therapeutic vulnerability in mismatch repair deficient cancer. Cancer cell, 37(3), 371-386.e12.
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4

Antibody Panel for Protein Analysis

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The following antibodies were used: SPOP (ab137537; Abcam), SPOP (16750-1-AP; proteintech), BRD2 (A302-583A; Bethyl), BRD2 (ab139690; Abcam), BRD3 (A302-368A; Bethyl), BRD4 (ab128874; Abcam), BRD4 (A301-985A; Bethyl), Myc (9E10; Sigma-Aldrich), Myc (SC-40; Santa Cruz Biotechnology), FLAG (M2; Sigma), HA (MM5-101R; Convance), Actin (AC-74; Sigma-Aldrich), DEK (13962S; Cell Signaling Technology), ERG (SC-352; Santa Cruz Biotechnology), AR (SC-816; Santa Cruz Biotechnology), SRC-3 (611104; BD), phospho-AKT-S473 (9471; Cell Signaling Technology), phospho-AKT-T308 (9275S; Cell Signaling Technology), AKT (9272; Cell Signaling Technology), phospho-S6K-T389 (9205; Cell Signaling Technology), S6K (9202; Cell Signaling Technology), β-tubulin (T4026; Sigma-Aldrich), RAC1 (23A8; BD), FDFT1 (ab195046; Abcam), DHCR24 (ab137845; Abcam), DHCR7 (ab103296; Abcam), MVD (ab12906; Abcam), HER3 (12708S; Cell Signaling Technology), INSR (ab131238; Abcam), IGF1R (SC-9038; Santa Cruz Biotechnology), mTOR (2972, Cell Signaling Technology ), Raptor (24C12, Cell Signaling Technology). MG132 and cycloheximide were purchased from Sigma-Aldrich, MLN4924, Bortezomib and MK2206 were purchased from Selleckchem. JQ1 was kindly provided by Dr. James Bradner and purchased from Sigma-Aldrich. i-BET762 (i-BET) was purchased from MedchemExpress. GDC-0068 was purchased from Calbiochem.
Zhang P., Wang D., Zhao Y., Ren S., Gao K., Ye Z., Wang S., Pan C.W., Zhu Y., Yan Y., Yang Y., Wu D., He Y., Zhang J., Lu D., Liu X., Yu L., Zhao S., Li Y., Lin D., Wang Y., Wang L., Chen Y., Sun Y., Wang C, & Huang H. (2017). Intrinsic BET inhibitor resistance in prostate cancer caused by SPOP mutation-mediated BET protein stabilization. Nature medicine, 23(9), 1055-1062.
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5

Investigating Mechanisms of Cell Cycle Regulation

Cited 1 time
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Cells were treated with 5 nM ActDL (Sigma-Aldrich, A1410) or 2 μM BMH-21 (Sigma-Aldrich, SML1183) for 24 hours. CHX (Sigma-Aldrich, C4859) was used at a working concentration of 100 μg/ml for 30 min or the indicated time points. For DOX induction (Sigma-Aldrich, D9891), cells were treated with DOX (1 μg/ml; Sigma-Aldrich, D9881) for 48 to 72 hours, unless otherwise indicated. Pan-caspase inhibitor Z-VAD-FMK (R&D Systems, FMK001) and caspase 2 inhibitor Z-VDVAD-FMK (R&D Systems, FMK003) were used at a final concentration of 20 μM for 1 hour or the indicated time points. MG132 (Sigma-Aldrich, M7449), Nutlin-3 (Selleckchem, S1061), and MLN4924 (Selleckchem, S7109) were added to cells in a final concentration of 10, 5, and 1 μM, respectively, for 24 hours. Camptothecin (Sigma-Aldrich, C9911) was used at a working concentration of 5 μM and 5-fluorouracil (Sigma-Aldrich, F6627) at 10 μg/ml. Neocarzinostatin was used at 0.5 μg/ml for 24 hours, LMB was used at 20 ng/ml for 6 hours, and pladB was used at 1 μM for 6 hours. For chemical inhibition of eIF4A3, 52a, or 52b, 53a from (31 (link)) was used at 5 μM for 24 hours. For cell synchronization at G2, cells were treated with nocodazole (Sigma-Aldrich, M1404) at 400 nM for 16 hours.
Kanellis D.C., Espinoza J.A., Zisi A., Sakkas E., Bartkova J., Katsori A.M., Boström J., Dyrskjøt L., Broholm H., Altun M., Elsässer S.J., Lindström M.S, & Bartek J. (2021). The exon-junction complex helicase eIF4A3 controls cell fate via coordinated regulation of ribosome biogenesis and translational output. Science Advances, 7(32), eabf7561.
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6

Preparation of Drug Stock Solutions

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MLN4924 and bortezomib for in vitro studies were purchased from Selleck Chemical (Houston, TX, USA). Stock solutions of each drug (10 mM) were made in 100% dimethyl sulfoxide (DMSO) and stored at −20 °C until use.
Han S., Shin H., Oh J.W., Oh Y.J., Her N.G, & Nam D.H. (2019). The Protein Neddylation Inhibitor MLN4924 Suppresses Patient-Derived Glioblastoma Cells via Inhibition of ERK and AKT Signaling. Cancers, 11(12), 1849.
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7

Antibody Sources and Chemical Reagents

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Antibodies against Beta-ACTIN (C-4), BACH1 (F-9) and LAMIN B2 (C-20) were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). Anti-NRF2 (D1Z9C) was obtained from Cell Signalling Technology (Danvers, MA, USA) and anti-HMOX1 was purchased from Biovision (San Francisco, CA, USA). Antibody against ALPHA-TUBULIN was obtained from Sigma-Aldrich (St. Louis, MO, USA). HRP-conjugated secondary antibodies were obtained from Life Technologies (Carlsbad, California, USA). Dimethyl sulfoxide (DMSO) was from Sigma-Aldrich. R,S-sulforaphane (SFN) was purchased from LKT Laboratories (St. Paul, MN, USA). (±)-TBE-31 was synthesized as described [41 ,42 (link)]. CDDO and CDDO-derivatives were obtained from Cayman Chemicals (Ann Arbor, MI, USA). MG132 was obtained from Santa Cruz Biotechnology, Leptomycin B from Cayman Chemicals, MLN4924 and Selinexor (KPT-330) from Selleckchem (Houston, TX, USA) and Actinomycin D and Cycloheximide from Sigma.
Casares L., Moreno R., Ali K.X., Higgins M., Dayalan Naidu S., Neill G., Cassin L., Kiib A.E., Svenningsen E.B., Minassi A., Honda T., Poulsen T.B., Wiel C., Sayin V.I., Dinkova-Kostova A.T., Olagnier D, & de la Vega L. (2022). The synthetic triterpenoids CDDO-TFEA and CDDO-Me, but not CDDO, promote nuclear exclusion of BACH1 impairing its activity. Redox Biology, 51, 102291.
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8

Cell Viability Assay with MLN4924

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Cell lines were treated with different concentrations of MLN4924 (Selleck Chemicals), MLN7243 (Chemie Tek), bortezomib (Selleck Chemicals), or a DMSO vehicle control in technical duplicates. After 3 days, viability assays were performed using PrestoBlue (Invitrogen) and a Tecan plate reader. After subtracting the media blank, viability was calculated following normalization to the DMSO vehicle control. Values represent an average of 3 independent experiments.
McGrail D.J., Garnett J., Yin J., Dai H., Shih D.J., Lam T.N., Li Y., Sun C., Li Y., Schmandt R., Wu J.Y., Hu L., Liang Y., Peng G., Jonasch E., Menter D., Yates M.S., Kopetz S., Lu K., Broaddus R., Mills G.B., Sahni N, & Lin S.Y. (2020). Proteome instability is a therapeutic vulnerability in mismatch repair deficient cancer. Cancer cell, 37(3), 371-386.e12.
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9

Dual Immunofluorescence Staining of Nedd8 and FLAG-Tagged Constructs

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For dual staining FLAG-tagged constructs and Nedd8, HCT-116 cells were transfected with desired constructs and treated with either DMSO, 1 μM MLN4924 (Selleck Chemicals), or 100 nM MLN7243 24 hours later, then incubated for an additional 24 hours. Cells were either directly fixed in neutral buffered formalin or pre-extracted in hypotonic stripping buffer (10mM HEPES, 1% Triton XI00, 10mM NaCl, 3mM MgC12, pH 7.4) prior to fixation. Cells were permabilized with 1% Triton X100 + 0.1% SDS, blocked in normal horse serum, and then stained overnight in anti-FLAG M2 (Sigma) and anti-Nedd8 (Cell Signaling) at 4°C. The following day, secondary antibodies anti-rabbit AlexaFluor 488 and anti-mouse AlexaFluor 594 were incubated for 1 hour at room temperature. For visualization of ubiquitin and Nedd8, the same procedure was followed except using HCT-116 cells stably expressing pEYFP-C3-HA-Ubiquitin and using anti-rabbit AlexaFluor 594 for detection of Nedd8. To quantify fraction of positive cells, a semi-automated MATLAB (R2019a) algorithm was utilized to automatically segment nuclei as described (McGrail et al., 2018b (link)), and then number of cells positive for BLVRA were counted manually for each image.
McGrail D.J., Garnett J., Yin J., Dai H., Shih D.J., Lam T.N., Li Y., Sun C., Li Y., Schmandt R., Wu J.Y., Hu L., Liang Y., Peng G., Jonasch E., Menter D., Yates M.S., Kopetz S., Lu K., Broaddus R., Mills G.B., Sahni N, & Lin S.Y. (2020). Proteome instability is a therapeutic vulnerability in mismatch repair deficient cancer. Cancer cell, 37(3), 371-386.e12.
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10

Cytotoxicity Screening of Anti-cancer Drugs

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Cells (5×105) were seeded in 6-well plates and allowed to adhere overnight. The following day, wells were treated with MLN4924 (Selleck Chemicals, 0.1 μM or 1.0 μM), camptothecin (10 μM) (Selleckchem), cisplatin (10 μM) (Selleckchem), a combination of 100 μM 5-fluorouracil (5-FU; Selleckchem) and 10 μM oxaliplatin (Selleckchem), or with the DMSO solvent control for 24 hours. Supernatants were harvested, cells cleared by centrifugation, and ATP quantified using the Invitrogen Molecular Probes ATP determination kit per the manufacturer’s instructions.
McGrail D.J., Garnett J., Yin J., Dai H., Shih D.J., Lam T.N., Li Y., Sun C., Li Y., Schmandt R., Wu J.Y., Hu L., Liang Y., Peng G., Jonasch E., Menter D., Yates M.S., Kopetz S., Lu K., Broaddus R., Mills G.B., Sahni N, & Lin S.Y. (2020). Proteome instability is a therapeutic vulnerability in mismatch repair deficient cancer. Cancer cell, 37(3), 371-386.e12.
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