Thalidomide (Toris Biosciences, 0652), pomalidomide (TCI chemicals, 19171-19-8), MLN4924 (Adooq BioScience, 905579-51-3), and all-trans retinoic acid (SIGMA, R2625) were purchased from the indicated vendors. Lenalidomide was a kind gift from Celgene.
Mln4924
Manufactured by Adooq
MLN4924 is a small molecule that inhibits the NEDD8-activating enzyme (NAE) complex. It functions by blocking the NEDD8 conjugation pathway, which is involved in the regulation of protein degradation and cell signaling processes.
Automatically generated - may contain errors
Lab products found in correlation
Aicar, by Merck Group (1 mentions)
D12345, by Thermo Fisher Scientific (1 mentions)
Trichostatin a, by Merck Group (1 mentions)
Resveratrol, by Selleck Chemicals (1 mentions)
L 755 507, by Bio-Techne (1 mentions)
Ni nta resin, by Thermo Fisher Scientific (1 mentions)
Phenylalanine, by Merck Group (1 mentions)
Methionine, by Merck Group (1 mentions)
Isoleucine, by Merck Group (1 mentions)
Lithium acetate, by Merck Group (1 mentions)
Yeast nitrogen base, by HiMedia (1 mentions)
On targetplus sirna, by Horizon Discovery (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Bromodeoxyuridine (brdu), by Abcam (1 mentions)
Gapdh g 9, by Santa Cruz Biotechnology (1 mentions)
Brca2, by Horizon Discovery (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Anti histone h3 phospho s10, by Abcam (1 mentions)
Brca1, by Horizon Discovery (1 mentions)
Rad51, by Abcam (1 mentions)
4 protocols using mln4924
1
Oncology Drug Screening Protocol
2
Small Molecule Compound Preparation
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Commercially available small molecules used in this study were NU7026 (SIGMA, T8552), Trichostatin A (SIGMA, T8552), MLN4924 (Adooq BioScience, A11260), NSC 19630 (Calbiochem, 681647), NSC 15520 (ChemBridge, 6048069), AICAR (SIGMA, A9978), RS-1 (Calbiochem, 553510), Resveratrol (Selleckchem, S1396), SCR7 (XcessBio, M60082-2s), L755507 (TOCRIS, 2197), B02 (SIGMA, SML0364), and STL127685 (Vitas-M). STL127685 is a 4-fluorophenyl analog of the non-commercially available STL127705. Stocks of 15 mM (or 10 mM for NU7026) were made using dimethylsulfoxide (DMSO) (Thermo Scientific, D12345). Solubility is a limiting factor for NU7026 concentration. Suitable working solutions for different concentrations were made so that addition of each small molecule accounts for a final concentration of 0.08% (or 0.2% for NU7026) DMSO in the media. Addition of all small molecules would lead to a final concentration of 0.7% DMSO.
3
Yeast Protein Expression Protocol
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Reagent Resource Centre (MR4). MLN4924 was from Adooq Bioscience, and all other biochemicals were from Sigma or Serva unless otherwise mentioned. Plasmid and genomic DNA isolation kits were from MACHEREY-NAGEL, cell culture reagents were from Lonza and Invitrogen, restriction enzymes and DNA modifying enzymes were from New England Biolabs and Thermo scientific. Antibodies were from Cell Signaling Technologies, Santacruz Biotechnology and Thermo Fisher Scientific. PCR reagents were from Thermo Fisher Scientific or New England Biolabs. Ni-NTA resin was from Thermo Fisher Scientific. Human blood was collected from healthy volunteers after written consent under medical supervision at the medical dispensary of the institute according to the protocols approved by the Institutional Ethical Committee of Centre for Cellular and Molecular Biology, India. Yeast extract, peptone, agar and yeast nitrogen base were from HiMedia. Glucose, galactose and amino acids (arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan) were from Sigma. Geneticin (G418) was from Calbiochem. Lithium acetate and PEG were from Sigma and the carrier DNA was from Gibco. S. cerevisiae strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0; Source EUROSCARF: FY1769) and plasmid pLE124 were kind gifts from Dr. Palani Murugan Rangasamy.
4
DNA Damage Response Pathway Analysis
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Reagents were obtained from MLN4924 (Adooq Biosciences), and cisplatin, Propidium iodide, RNase and BrdU (Sigma-Aldrich). Anti-y-H2AX (S139), pATR (S428), pATM (S1981), pChk1 (S345), pChk2 (T68) are from DNA damage antibody sampler kit from Cell Signaling Technology. Chk1, Chk2, Cdt1 (D10F11), p21, BRCA2 (D9S6V), and cleaved caspase 3 (D175) from Cell Signaling Technology. p27, Cdc25A (F-6), BRCA1, ATR (N-19), ATM, β-Actin, and GAPDH (G-9) are from Santa Cruz Biotechnology. APPBP1/NAE1, UBE1C/UBA3, Rad51, BrdU, and Anti-histone H3 phospho S10, are from Abcam. HRP conjugated Anti-mouse, Anti-rabbit, and Anti-goat secondary antibodies were obtained from GE Healthcare UK Limited. Fluorescent conjugated (Texas red and FITC) secondary antibodies were from Santa Cruz Biotechnology. BRCA1, BRCA2, and control siRNAs were from Dharmacon (ON-TARGET Plus siRNA, Dharmacon).
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