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Cycloheximide (chx)

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Cycloheximide is a laboratory reagent used to inhibit protein synthesis in eukaryotic cells. It acts by blocking the translocation step in protein synthesis, preventing the translation of mRNA into proteins.

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Lab products found in correlation

Mg132, by Merck Group (9 mentions) Mg132, by Cell Signaling Technology (5 mentions) Dulbecco s modified eagle medium, by Thermo Fisher Scientific (4 mentions) Mg132, by Selleck Chemicals (4 mentions) Celltiter glo, by Promega (3 mentions) Z vad fmk, by Selleck Chemicals (3 mentions) Co2 incubator, by Thermo Fisher Scientific (3 mentions) Cycloheximide (chx), by R&D Systems (2 mentions) Mg132, by R&D Systems (2 mentions) Mln4924, by R&D Systems (2 mentions) Mg132, by Thermo Fisher Scientific (2 mentions) Actinomycin d, by Merck Group (2 mentions) Tnf α, by R&D Systems (2 mentions) Leupeptin, by Merck Group (2 mentions) Digoxin, by Merck Group (2 mentions) L glucose, by Merck Group (2 mentions) Hypoxia chamber, by Coy Laboratory Products (2 mentions) D glucose, by Merck Group (2 mentions) Hoechst 33342 solution, by Thermo Fisher Scientific (2 mentions) Ifn γ, by Thermo Fisher Scientific (2 mentions)

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Cycloheximide (CHX) (#2112s) (3 citations)

74 protocols using cycloheximide (chx)

1

Investigating HER3 Downregulation Mechanisms

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To examine HER3 levels, cells were treated with the indicated concentrations of affibody, pan-HER kinase inhibitor (Millipore 324840), or no treatment (media alone) control, lysed at the indicated time points, and probed by immunoblot for HER3 as previously described using primary antibodies (D22C5 HER3 and 13E5 β-actin). To investigate the mechanism of HER3 downregulation, a cycloheximide pulse-chase experiment was performed where cells were treated with 10 nM affibodies, 178 μM cycloheximide (Cell Signaling 2112), or the combination, lysed at the indicated time points, and probed by immunoblot. For HER3 degradation studies, cells were pretreated with 200 μM leupeptin (Sigma) or 5 μM MG-132 (Sigma) for 3 h, treated with 10 nM affibodies, 200 μM leupeptin (Sigma), 5 μM MG-132 (Sigma), or combinations of affibody plus inhibitor, lysed at the indicated time points, and probed by immunoblot.
Schardt J.S., Oubaid J.M., Williams S.C., Howard J.L., Aloimonos C.M., Bookstaver M.L., Lamichhane T.N., Sokic S., Liyasova M.S., O’Neill M., Andresson T., Hussain A., Lipkowitz S, & Jay S.M. (2017). Engineered Multivalency Enhances Affibody-Based HER3 Inhibition and Downregulation in Cancer Cells. Molecular pharmaceutics, 14(4), 1047-1056.
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2

Measuring Oct-1 Protein Stability with CHX

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Oct-1 protein stability was measured on treatment with protein synthesis inhibitor cycloheximide (CHX). Cells treated with 100 μg ml−1 CHX (#2112, Cell Signaling Technology, Inc.) were collected at different time points and cell lysate was used for western blot to determine the protein level at different CHX treatment time. Western blot results were quantified by the ImageJ Software (NIH). Three independent experiments were performed and decay curve was plotted.
Du L., Li Y.J., Fakih M., Wiatrek R.L., Duldulao M., Chen Z., Chu P., Garcia-Aguilar J, & Chen Y. (2016). Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal. Nature Communications, 7, 12326.
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3

Measuring c-Myc Protein Stability

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c-Myc protein stability was measured on treatment with protein synthesis inhibitor cycloheximide (CHX). Cells treated with 100 μg ml− 1 CHX (#2112, Cell Signaling Technology) were collected at different time points and cell lysate was used for western blot to determine the protein level at different CHX treatment time. Western blot results were quantified by the ImageJ Software (NIH).
Du L., Liu W., Aldana-Masangkay G., Pozhitkov A., Pichiorri F., Chen Y, & Rosen S.T. (2022). SUMOylation inhibition enhances dexamethasone sensitivity in multiple myeloma. Journal of Experimental & Clinical Cancer Research : CR, 41, 8.
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4

Cycloheximide-based Protein Stability Assay

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IRF4 protein stability was measured on treatment with protein synthesis inhibitor cycloheximide (CHX). Cells treated with 100 μg ml−1 CHX (#2112, Cell Signaling Technology, Inc.) were collected at different time points and cell lysate was used for western blot to determine the protein level at different CHX treatment time. Western blot results were quantified by the ImageJ Software (NIH).
Du L., Liu W., Pichiorri F, & Rosen S.T. (2022). SUMOylation inhibition enhances multiple myeloma sensitivity to lenalidomide. Cancer Gene Therapy, 30(4), 567-574.
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5

Protein Turnover Analysis in Isogenic Cell Lines

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HCT116, DNMT1KO-HCT116 and CTNNB1KO-HCT116 were grown in separate 6-well plates to log phase. Cycloheximide (Cell Signaling 2112, Danvers, MA) was added to a final concentration of 50 μg/ml to terminate protein synthesis. After Cycloheximide treatment, equal numbers of cells were collected at 0, 3, and 6 hours, and cell lysates were prepared as described. The lysates were analysed by SDS-PAGE and Western blotting. Bands corresponding to each protein were detected by using chemiluminescence and the intensity of bands was quantified using ImageJ. Half-life (T1/2) was calculated as previously described (26 (link)).
Song J., Du Z., Ravasz M., Dong B., Wang Z, & Ewing R.M. (2015). A protein interaction between β-catenin and Dnmt1 regulates Wnt Signaling and DNA methylation in colorectal cancer cells. Molecular cancer research : MCR, 13(6), 969-981.
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6

Cycloheximide-Induced Protein Degradation

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TA-DC were purified from tumors as described above and treated with 50μg/ml cycloheximide (Cell Signaling) for 0 – 24 hours in a 24 well dish. Phosphorylated FOXO3 found in the cytoplasm can be rapidly degraded, therefore the initial time points 0, 5, 10, 15 and 30 min were the primary focus of western blot analysis. STAT3 degradation was observed to degrade in response to cycloheximide treatment and was therefore used as a control that protein degradation did occur in upon treatment.
Thompson M.G., Larson M., Vidrine A., Barrios K., Navarro F., Meyers K., Simms P., Prajapati K., Chitsike L., Hellman L.M., Baker B.M, & Watkins S.K. (2015). FOXO3–NF-κB RelA Protein Complexes Reduce Proinflammatory Cell Signaling and Function. The Journal of Immunology Author Choice, 195(12), 5637-5647.
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7

Measuring GPC1 Protein Stability

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Targeted cells were seeded in 6-well plates and incubated in a CO2 incubator overnight. After 12-hrs incubation, the medium was removed and replenished with a complete medium with 20 μg/ml cycloheximide (Cell Signaling Technology, #2112). Cell lysates were collected at different time points (t=0, 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, and 24 hrs) according to the experimental design. Cell lysates were centrifuged at 12,000 rpm at 4 oC for 30 mins. Protein concentration was determined by the BCA protein assay kit (Pierce). The results were then analyzed by SDS-PAGE assay and Western blotting. The Western bands of GPC1 and β-actin were quantified using Image Lab Software (BioRad).
Pan J., Li N., Renn A., Zhu H., Chen L., Shen M., Hall M.D., Qian M., Pastan I, & Ho M. (2022). GPC1-targeted immunotoxins inhibit pancreatic tumor growth in mice via depletion of short-lived GPC1 and downregulation of Wnt signaling. Molecular cancer therapeutics, 21(6), 960-973.
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8

Investigating Protein Turnover Regulators

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HeLa cells at 80% confluence were transfected using Lipofectamine 2000 with pLentiLox-hH1 vectors containing either a specific sequence targeting GlyRS, SerRS or the vector alone. Forty-eight hours after transfection, medium was replaced with that containing 30 μg/mL cycloheximide (#2112; Cell signalling) or 20 μM MG-132 (#508338, Fisher). MLN4924 (I-502; Boston-biochem) samples were prepared by treating the cells with 0.2 μM MLN4924 for 24 h and then followed by cycloheximide or MG132 treatment. Samples were collected and lysed with acidic lysis buffer and later subjected to SDS-PAGE.
Mo Z., Zhang Q., Liu Z., Lauer J., Shi Y., Sun L., Griffin P.R, & Yang X.L. (2016). Neddylation requires glycyl-tRNA synthetase to protect activated E2. Nature structural & molecular biology, 23(8), 730-737.
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9

Evaluating USP15 Regulation on HBx

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0.75 μg of pHBx-FLAG were co-transfected into Huh7 cells with 2.25 μg of pUSP15-myc or 40 pmol of USP15 siRNA. The equal mole amounts of pcDNA3.1/myc-His(−)A or control siRNA were used as a negative control. 48 hours posttransfection, cells were treated with 100 μg/ml of cycloheximide (Cell Signaling Technology) at indicated times. Cells were washed three times with prechilled PBS and The cells were lysed with RIPA lysis buffer (Pierce) containing a proteinase inhibitor cocktail (Roche Diagnostics) and 1 mM PMSF, and followed by SDS-PAGE and Western blot analysis.
Su Z.J., Cao J.S., Wu Y.F., Chen W.N., Lin X., Wu Y.L, & Lin X. (2017). Deubiquitylation of hepatitis B virus X protein (HBx) by ubiquitin-specific peptidase 15 (USP15) increases HBx stability and its transactivation activity. Scientific Reports, 7, 40246.
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10

Protein Stability Analysis via Cycloheximide

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The proteins were expressed for 24h and then serum starved for an additional 24h. Then, cells were treated with 100 μg/mL cycloheximide (Cell Signaling, cat# 2112). At the indicated times, 100 μl of 2X SDS-PAGE sample buffer was added and the cells were scraped from the wells, and boiled for 5 min. After all lysates were collected, each sample was loaded onto a 10% SDS-PAGE gel and then analyzed by western blotting.
Ivanov A.A., Gonzalez-Pecchi V., Khuri L.F., Niu Q., Wang Y., Xu Y., Bai Y., Mo X.L., Prochownik E.V., Johns M.A., Du Y., Khuri F.R, & Fu H. (2017). OncoPPi-informed discovery of Mitogen-Activated Protein Kinase Kinase 3 as a novel binding partner of c-Myc. Oncogene, 36(42), 5852-5860.
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