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Cycloheximide c7698

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Cycloheximide (C7698) is a chemical compound commonly used in laboratory settings. It functions as a protein synthesis inhibitor, specifically targeting the 60S subunit of eukaryotic ribosomes. This mechanism of action makes Cycloheximide a valuable tool for researchers studying cellular processes that involve protein synthesis.

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Lab products found in correlation

Mg132 c2211, by Merck Group (2 mentions) Mg132 m7449, by Merck Group (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Chymotrypsin, by Promega (1 mentions) Ab87230, by Abcam (1 mentions) Ab108414, by Abcam (1 mentions) Cell counting kit 8 ck04, by Dojindo Laboratories (1 mentions) Pfu turbo polymerase, by Agilent Technologies (1 mentions) Ab175932, by Abcam (1 mentions) Ab252432, by Abcam (1 mentions) Ab106814, by Abcam (1 mentions) Ab203912, by Abcam (1 mentions) Ab280888, by Abcam (1 mentions) Ab40855, by Abcam (1 mentions) Pa5 109993, by Thermo Fisher Scientific (1 mentions) Ab32042, by Abcam (1 mentions) 5 aza 2 deoxycytidine a3656, by Merck Group (1 mentions) Mda mb 231, by American Type Culture Collection (1 mentions) L glutamine, by Lonza (1 mentions) Ab168372, by Abcam (1 mentions)

Close spelling variants of this product

Cycloheximide (C7698-5G) C7698

11 protocols using cycloheximide c7698

1

Akt Isoforms Regulation by Calpain-1

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Human apoE3 (SRP4696), Calpain-1 (C6108) and cycloheximide (C7698) were purchased from Sigma-Aldrich (St. Louis, MO). Lipofectamine 2000 (#11668-027), Trizol (15596-026), siRNAs for control (#AM4611), Akt1 (#AM1670), Akt2 (#AM16708), and Akt3 (#AM16708) were purchased from Life Technologies (Carlbad, CA). Expression plasmids for Akt1 (EX-A0022-M02) and Akt2 (EX-X0037-M02) were purchased from Genecopoeia (Rockville, MD). Pepstatin A (sc-45036), lactacystin (sc-3575), ALLN (sc-221136), batimastat (sc-203833), leupeptin-HCl (sc-215242), aprotinin (sc-3595), Akt inhibitor X (sc-203811), Akt inhibitor XI (sc-221229), PI3K inhibitor LY294002, and antibodies against ABCA1 (sc-58219), β-actin (sc-47778), GAPDH (sc-32233), CD-MPR (sc-34573), p-Akt (sc-7985-R), Akt (sc-8312), and Sp1 (sc-14027) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against p-Sp1 (T739) was purchased from Assay Biotechnology (Sunnyvale, CA). Antibodies against Akt isoforms (#9940S), pAkt1 (#9018S), and pAkt2 (#8599S) were purchased from Cell Signaling Technology (Danvers, MA). Antibody against pAkt3 (#bs-5209R) was purchased from One World Lab (San Diego, CA). Calpain activity kit (#4240-100) was purchased from Biovision (Milpitas, CA).
Okoro E.U., Guo Z, & Yang H. (2016). Akt Isoform-Dependent Regulation of ATP-Binding Cassette A1 Expression by Apolipoprotein E. Biochemical and biophysical research communications, 477(1), 123-128.
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2

Protein Purification and Antibody Validation

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Antibodies used in this study were principally purchased from Santa Cruz Biotechnology (G6PD (G-12): SC-373886 (dilution 1:1000), 6PGD (G-2): SC-398977 (dilution 1:1000), His (HIS.H8): SC-57598 (dilution 1:1000), Enolase (H-300): SC-15343 (dilution 1:1000)), Cell Signaling Technology (beta actin (8H10D10): 3700 S (dilution 1:1000)), Everest Biotech (G6PD: EB07841 (dilution 1:1000)), Advance Immunochemical (GAPDH (6C5): 6C5 (dilution 1:2000)), and Abcam (G6PD: AB87230 (dilution 1:1000), band 3 (EPR1426): AB108414 (dilution 1:1000)). TALON Superflow (28-9575-02) and bovine thrombin (91-030) were purchased from GE Healthcare and BioPharma for protein purification. Pfu Turbo polymerase (600252) used for site-directed mutagenesis was purchased from Agilent Technologies. Chymotrypsin was purchased from Promega (V1061). Cell counting kit-8 (CK04) and glutathione quantification kit (T419) were purchased from Dojindo. Cycloheximide (C7698) and chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (CM-H2DCFDA, C6827) were purchased from Sigma and Thermo Fisher Scientific, respectively. Glutathione assay kit (703002) used for blood assays was purchased from Cayman Chemical.
Hwang S., Mruk K., Rahighi S., Raub A.G., Chen C.H., Dorn L.E., Horikoshi N., Wakatsuki S., Chen J.K, & Mochly-Rosen D. (2018). Correcting glucose-6-phosphate dehydrogenase deficiency with a small-molecule activator. Nature Communications, 9, 4045.
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3

Astragaloside IV Antioxidant Mechanism

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Astragaloside IV (C41H68O14, A800922, purity ≥98.5%) was purchased from Maclin Inc. and the chemical structure is shown as Figure 1A. ROS fluorescent probe‐DHE (R001, Vigorous Biotechnology) used for detection of ROS in tissues and DCFH‐DA (KM0062) used for detection of cellular ROS was purchased from Beijing Biolab Technology Co. The antibodies used in this study including NRF1 (ab175932, abcam), PGC‐1α (ab106814, abcam), TFAM (ab252432, abcam), Caspase3 (9662, CST), cleaved‐caspase3 (ab32042, abcam), Bax (ab111391, abcam), Bcl2 (2872, CST), α‐SMA (A5228, sigma), p‐smad2 (ab280888, abcam), smad2 (ab40855, abcam), MT‐CO1 (ab203912, abcam), MT‐ND6 (PA5‐109993, Invitrogen) and MT‐ATP6 (PA5‐116789, Invitrogen). MG‐132 (M7749) and Cycloheximide (C7698) were purchased from Sigma.
Xie M., Xia B., Xiao L., Yang D., Li Z., Wang H., Wang X., Zhang X, & Peng Q. (2023). Astragaloside IV ameliorates peritoneal fibrosis by promoting PGC‐1α to reduce apoptosis in vitro and in vivo. Journal of Cellular and Molecular Medicine, 27(19), 2945-2955.
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4

Culturing TNBC Cell Lines

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MDA-MB-231 and Hs578T TNBC breast cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza) containing 10% fetal bovine serum (FBS, ThermoFisher Scientific) and 2 mM L-glutamine (Lonza), unless otherwise specified. L-carnosine (C9625), aminoguanidine (396,494), methylglyoxal (MG, M0252) and 5’Aza-2’-Deoxycytidine (A3656) and cycloheximide (C7698) were from Sigma.
Dube G., Tiamiou A., Bizet M., Boumahd Y., Gasmi I., Crake R., Bellier J., Nokin M.J., Calonne E., Deplus R., Wissocq T., Peulen O., Castronovo V., Fuks F, & Bellahcène A. (2023). Methylglyoxal: a novel upstream regulator of DNA methylation. Journal of Experimental & Clinical Cancer Research : CR, 42, 78.
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5

Western Blot and IP Antibodies

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The following antibodies were used in western blot: anti-FBXO32 (NBP1-36955, Novus, Littleton, CO, USA); anti-FBXO32 (ab168372, Abcam, Cambridge, MA, USA); anti-KLF4 (sc-20691, Santa-Cruz, Dallas, TX, USA); anti-KLF4 (ab75486, Abcam); anti-ubiquitin (3936, CST, Billerica, MA, USA); anti-HA (sc-805, Santa-Cruz); anti-GST (G7781, Sigma, St Louis, MO, USA); anti-Flag (F7425, Sigma), anti-Myc (562, MBL, Woburn, MA, USA). The following antibodies were used in immunohistochemistry and immunofluorescent double staining: anti-FBXO32 (LS-B9748, Lifespan Biosciences, Seattle, WA, USA) and anti-KLF4 (NBP2-24749, Novus). The anti-KLF4 antibody (12173, CST) and anti-FLAG M2 Affinity Gel (A2220, Sigma) were used for IP. The Glutathione Sepharose 4B (17-0756-01, GE Healthcare, Amersham, UK) was used in GST pulldown. Cycloheximide (C7698) and MG132 (C2211) were purchased from Sigma. SB203580 was obtained from Promega (Madison, WI, USA) and SB239063 was purchased from Selleckchem (Houston, TX, USA).
Zhou H., Liu Y., Zhu R., Ding F., Wan Y., Li Y, & Liu Z. (2017). FBXO32 suppresses breast cancer tumorigenesis through targeting KLF4 to proteasomal degradation. Oncogene, 36(23), 3312-3321.
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6

Antibody and Kinase Inhibitor Protocol

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The following antibodies were used, from Cell Signaling Technology: anti-MYC (5605), anti-Pol II (14958), anti-pSTAT1Ser727 (8826), anti-STAT1 (14995), anti-pERK (4370), anti-ERK (9201), anti-CDK9 (2316); from AbCam: anti-pMYC Ser62 (ab106952), anti-pPol II Ser2 (ab5095); from Sigma-Aldrich: anti-KRAS (WH0003845M1), anti-vinculin (V9131) from Santa Cruz Biotechnology: anti-MCL1 (sc-12756). The protein kinase inhibitors NVP-2 (HY-12214A), amuvatinib (HY-10206) and imatinib (HY-15463) were obtained from MedChemExpress, TC-S 7004 (5088) from Tocris Bioscience, and AZ 191 (5232) from Sigma-Aldrich. MG132 (M7449) and cycloheximide (C7698) were also obtained from Sigma-Aldrich. THAL-SNS-032 was a gift from Dr. Nathanael Gray (Harvard/DFCI). Synthetic schemes for UNC10112731 and UNC10112785 and derivatives are shown in Supplemental Methods.
Blake D.R., Vaseva A.V., Hodge R.G., Kline M.P., Gilbert T.S., Tyagi V., Huang D., Whiten G.C., Larson J.E., Wang X., Pearce K.H., Herring L.E., Graves L.M., Frye S.V., Emanuele M.J., Cox A.D, & Der C.J. (2019). Application of a MYC degradation screen identifies sensitivity to CDK9 inhibitors in KRAS-mutant pancreatic cancer. Science signaling, 12(590), eaav7259.
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7

Reagents for Cellular Stress Signaling

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The following reagents were used in our study: collagen (Roche, Indianapolis, IN; 11179179001), G418 (Invitrogen, Carlsbad, CA), recombinant human TNFα (PeproTech, Rocky Hill, NJ; 300-01A), anisomycin (Fermentek, Jerusalem, Israel; AN), arsenite (Fluka, Ronkonkoma, NY; 71287). Sorbitol (S1876), sodium palmitate (P9767), puromycin (P7255), 5Z-7-Oxozeaenol (O9890), and cycloheximide (C7698) were all purchased from Sigma-Aldrich (St. Louis, MO). URMC-099 was purchased from AdooQ BioScience LLC (Irvine, CA; A14219). Takinib was purchased from MedChemExpress (Monmouth Junction, NJ; HY-103490). CEP-1347 was purchased from Tocris Bioscience (Bristol, UK; 4924).
Prinz E., Aviram S, & Aronheim A. (2018). WDR62 mediates TNFα-dependent JNK activation via TRAF2-MLK3 axis. Molecular Biology of the Cell, 29(20), 2470-2480.
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8

Cell Line Cultivation and Treatments

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U2OS, HEK293T, A549, and H1299 cell lines were obtained from ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (01‐055‐1A) supplemented with 10% fetal bovine serum (04‐007‐1A), 100 U·mL−1 penicillin, and 0.1 mg·mL−1 streptomycin sulfate (03‐031‐1B) and 2 mml‐glutamine (03‐020‐1B) from Biological Industries (Beit HaEmek, Israel). Cells were treated where indicated with 20 μg·mL−1 cycloheximide (C7698), 10 μg·mL−1 (0.015 U·mL−1) bleomycin sulfate (B5507) or 10 μm MG132 (C2211) from Sigma‐Aldrich/Merck (Darmstadt, Germany).
García‐Cano J., Sánchez‐Tena S., Sala‐Gaston J., Figueras A., Viñals F., Bartrons R., Ventura F, & Rosa J.L. (2019). Regulation of the MDM2‐p53 pathway by the ubiquitin ligase HERC2. Molecular Oncology, 14(1), 69-86.
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9

Immunofluorescence detection of stress granules

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The rabbit polyclonal anti-P antibody was previously described [23 (link)]. The mouse monoclonal anti-G3BP-1 (2 F3) antibody was obtained from Sigma. The rabbit polyclonal anti-P antibody was previously described [23 (link)]. The rabbit anti-phospho eIF2α (04342) was obtained from Millipore. The Rabbit anti-MDA5 (33H12L34) was from Invitrogen, the rabbit anti-RIG-I (AT111) was from Enzo life; the goat monoclonal anti-TIA-1 (C20) and mouse anti-PABP (10E10) antibodies were from Santa-Cruz Biotechnology. Secondary fluorescent antibodies were purchased from Molecular Probes (Alexa fluor 488-, 568- or 647- conjugated) and Cell Signaling (Fluor 800 or Fluor 680 conjugated). Nocodazole (M1404), actinomycin D (A9415), sodiumarsenite (S7400) and cycloheximide (C7698) were obtained from Sigma.
Nikolic J., Civas A., Lama Z., Lagaudrière-Gesbert C, & Blondel D. (2016). Rabies Virus Infection Induces the Formation of Stress Granules Closely Connected to the Viral Factories. PLoS Pathogens, 12(10), e1005942.
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10

Protein Interactome Investigation Protocol

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MG132 (M7449), Cycloheximide (C7698), and Hexadimethrine bromide (H9268) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal antibodies against Flag (Sigma-Aldrich, St. Louis, MO, USA), HA (Promotor Biotechnology, Wuhan, China), and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal antibodies against HA (Sigma-Aldrich); rabbit anti-ERK1/2 and p-ERK1/2 (Cell Signaling Technology, Danvers, USA); rabbit anti-USP21 and rabbit anti-MEK2 (Proteintech Biotechnology, Wuhan, China). Anti-Flag affinity agarose, anti-HA affinity agarose (Selleck, Houston, USA).
Li W., Cui K., Prochownik E.V, & Li Y. (2018). The deubiquitinase USP21 stabilizes MEK2 to promote tumor growth. Cell Death & Disease, 9(5), 482.
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