HepG2-HBx-FSH8 cells were induced with 120 ng/ml doxycycline for 24 hours, treated with MLN4924 (1µM, Cayman Chemical) or DMSO for 4 hours, and lysed in NP-40 Lysis Buffer (0.5% Nonidet P-40, 50 mM Tris pH 7.5, 150 mM NaCl) with Halt protease inhibitor (Pierce) and DMSO or MLN4924 on ice after culturing. Flag-SBP-HBx was precipitated with anti-Flag M2 resin (Sigma) affinity eluted with 200µg/ml 3x FLAG peptide (Sigma). Eluates were then incubated with streptavidin agarose (GE Life Sciences). Beads were washed 4 times with lysis buffer, and peptides were generated by on-beads trypsin digestion and isolated by filter-aided sample preparation (FASP). LCMS was performed as described (Yu and Pieper, 2015 (link)). Database search was conducted using SEQUEST and Proteome Discoverer (Yu et al., 2014 (link)). Spectral counts were compared between samples, and those proteins with ≥4 fold increase over negative control runs and increased after MLN4924 treatment were selected as potential HBx substrates. Anti-FLAG immunoprecipitations for IP western analysis were performed as above, but eluted at 70°C for 20 minutes in SDS-PAGE loading buffer.
Mln4924
Manufactured by Cayman Chemical
Sourced in United States
MLN4924 is a small molecule that inhibits the NEDD8-activating enzyme (NAE). It functions by blocking the activation and conjugation of the ubiquitin-like protein NEDD8 to its target proteins, which is an essential step in the cullin-RING E3 ubiquitin ligase (CRL) activation pathway.
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Lab products found in correlation
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (4 mentions)
Mg132, by Cayman Chemical (3 mentions)
Bortezomib, by Cayman Chemical (3 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Anti rabbit irdye 800cw, by LI COR (2 mentions)
Flag m2 monoclonal ab, by Merck Group (2 mentions)
Mtmr1, by Abcam (2 mentions)
D2hgdh, by Abcam (2 mentions)
Igbp1, by Cell Signaling Technology (2 mentions)
Smad9, by Abcam (2 mentions)
Actr5, by Proteintech (2 mentions)
Akap8, by Abcam (2 mentions)
Gstp1, by Cell Signaling Technology (2 mentions)
Anti mouse irdye 800cw, by LI COR (2 mentions)
Irdye 680cw anti rabbit, by LI COR (2 mentions)
Anti mouse irdye 680cw, by LI COR (2 mentions)
Me bs, by Merck Group (2 mentions)
Tmtsixplex isobaric label reagent, by Thermo Fisher Scientific (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Val boropro, by Merck Group (2 mentions)
22 protocols using mln4924
1
Identification of HBx Interacting Proteins
2
Protein Synthesis and Proteasome Inhibition
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To inhibit de novo protein synthesis, cells were treated with 50 μg/ml of CHX (CAS number 01810; Sigma). For proteasome inhibition, keratinocytes were treated with 20 μM MG132 (catalog number 133407-82-6; Cayman Chemical). To inhibit cullin neddylation, cells were treated with 33 nM to 3 μM MLN4924 (CAS number 905579-51-3; Cayman Chemical). Cell viability was determined using a CellTiter-Glo luminescent cell viability assay (Promega) according to the supplier's instructions.
3
Protein Kinase and Nedd8 Inhibitor Assay
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The protein kinase CKII inhibitor TBB (Tocris Bioscience) and the small-molecule inhibitor of Nedd8-activating enzyme, MLN4924 (Cayman Chemical), were prepared as 1 mM stocks in dimethyl sulfoxide (DMSO) and were stored at −20°C. TBB and MLN4924 were diluted in cell culture medium to final concentrations of 80 and 1 μM, respectively, immediately prior to use. TNF-α (Sigma) was dissolved in water at a concentration of 25 μg/ml and diluted in medium to a final concentration of 25 ng/ml. MG132 (Sigma) was dissolved in DMSO and diluted in medium to a final concentration of 15 μM.
4
Cell Lines and Virus Expansion Protocol
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HEK293T, BS-C-1, and RK-13 were grown in Dulbecco modified Eagle medium (DMEM) (Life Technologies) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Seralab), 100 U/ml penicillin, and 100 μg/ml streptomycin (Pen/Strep) (Life Technologies). HEK293T-REx cells were grown as described above with the addition of 10 μg/ml blasticidin (Life Technologies). ECTV strain Moscow was from Antonio Alcami and was expanded in BS-C-1 cells. CPXV strain Brighton Red was from Geoffrey L. Smith and was expanded in RK-13 cells. Both viruses were titrated by conventional plaque assay. SeV was a gift from Steve Goodbourn. IL-1β, TNF-α, and IFN-β were from Peprotech. PMA was from Santa Cruz Biotechnology. MLN4924 was from Cayman Chemical.
5
Culturing Hematological Cell Lines
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HL60, U937, NB4, and MM1S cells were cultured in RPMI medium (#10247302, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and 1% P/S. UM171 (#72914) and UM729 (#72332) were obtained from Stem cell technologies and dissolved in DMSO. MLN4924 (#15217) and Bortezomib (#10008822) were obtained from Cayman chemicals and the stocks were prepared in DMSO.
6
Antibody-Mediated Protein Interactome Analysis
7
Proteasome Inhibition and TRP Modulation
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The following chemicals were used: bortezomib, carfilzomib, AMG9810, capsaicin and MLN4924 from Cayman.
8
Proteasomal Regulation by FKB in Cancer
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FKB with 99% purity was isolated from kava extracts by LKT Laboratories, Inc. (St. Paul, MN). Bortezomib, MG-132 and MLN4924 were obtained from Cayman Chemical Inc. (Ann Arbor, MI). Antibodies against Ubc12, ubiquitin, and β-tubulin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-Skp2 antibodies were purchased from Invitrogen (Grand Island, NY). Anti-Myc-tag and Anti-cleaved-PARP antibodies were from Cell Signaling (Boston, MA). Anti-Cullin-1 and anti-NEDD8 antibodies were from Abcam (Cambridge, MA). Anti-p27/Kip1 and p21/WAF1 antibodies were from BD Biosciences (Billerica, MA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Sigma. The Reverse Transcription System kit and was from Promega (Mandison, WI). A quantitative reverse transcription polymerase chain reaction (RT-PCR) kit was from Bio-Rad (Hercules, CA). Ubiquitylation Assay Kit and 20S Proteasome Assay Kit were from Cayman and Abcam, respectively.
9
Inducible CDT1 expression in U2OS cells
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Human HCT1116 cells and U2OS cells were grown at 37 °C in a 5% CO2 atmosphere in Dulbecco’s modified Eagle medium (ThemoFisher, 10564011), supplemented with 10% fetal calf serum. MLN4924 was purchased from Cayman Chemicals (15217-1). CDT1 cDNA was obtained by reverse transcription and PCR with primers CDT1-F and CDT1-R (Supplementary Table 6 ) from HCT116 cells. CDT1 cDNA amplified by HindIII_Cdt1F and XhoI_Cdt1R primers (Supplementary Table 6 ) was inserted to pCMV-Tag2A (https://www.addgene.org/vector-database/6193/ ). CDT1 sequence was verified using sanger sequencing. CDT1 cDNA was further cloned into the Tet-On 3 G inducible expression system (TaKaRa, 631168) with a flag tag at the N terminal and an EGFP tag at the C terminal by the In-Fusion HD Cloning system (TaKaRa, 638909) with infusion primers CDT1F and CDT1R (Supplementary Table 6 ). After transfection of the U2OS cells with the pCMV-Tet3G Vector, stable clones were further transfected with the pTRE3G Vector containing flag-CDT1-GFP. Stable clones were tested for GFP positivity (CDT1-GFP) and re-replication by flow cytometry and presence of fusion protein by western blotting with anti-CDT1 antibody.
10
Monitoring Cell Cycle Progression
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HCT-116 colon cancer cells (ATCC CCL-247) were cultured in McCoy's 5A medium (Corning) supplemented with 10% fetal bovine serum (FBS). For mitotic arrest, nocodazole (100ng/mL) was added to the media for 4 h. Cells were collected by manual shake-off and washed four times in phosphate-buffered saline (PBS) before re-seeding in culture medium. Time points were taken at intervals after release to monitor cell cycle progression. Flp-In T-rex 293 cells (Invitrogen, R78007) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Corning) supplemented with 10% FBS. For transgene induction, doxycycline (2.5 ug/ml, Enzo) was added to the medium for 24 h. MG132 (20 uM, Selleck), MLN4924 (3 uM, Cayman Chemical), Palbociclib (100 nM, Selleck), T2AA (hydrochloride), (20 uM, Cayman Chemical), or NU-6102 (20 uM, Cayman Chemical) were added to the cell culture medium for the indicated times.
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