Cycloheximide (chx)

Manufactured by Merck Group

Sourced in United States, Germany, United Kingdom, China, Italy, Sao Tome and Principe, France, Macao, Japan, Spain, Canada, Switzerland, India, Israel, Brazil, Poland, Portugal, Australia, Morocco, Sweden, Austria, Senegal, Belgium

Cycloheximide is a laboratory reagent commonly used as a protein synthesis inhibitor. It functions by blocking translational elongation in eukaryotic cells, thereby inhibiting the production of new proteins. This compound is often utilized in research applications to study cellular processes and mechanisms related to protein synthesis.

Automatically generated - may contain errors

Lab products found in correlation

3 702 protocols using cycloheximide (chx)

Exploring β-catenin Protein Regulation

Check if the same lab product or an alternative is used in the 5 most similar protocols

CHX-chase assay was undertaken with CHX (Sigma-Aldrich, Darmstadt, Germany), serving as the protein synthesis inhibitor. In detail, 50 μg/ml of CHX mixed in serum-free medium was employed to treat the cells in six-well plates, and β-catenin protein was investigated through Western blot analysis at 0, 2, 4, and 6 h. Furthermore, 25 μM MG132 (Sigma-Aldrich, Darmstadt, Germany), the proteasome inhibitor, was used to rescue CHX-chase assay.
For ubiquitination assay, 293T cells were cotransfected with hemagglutinin (HA)-tagged ubiquitin vector (Ub-HA), myc-tagged β-catenin vector (β-catenin-myc), and LYPLAL1-DT overexpression vector or LYPLAL1-DT knockdown silencer, along with the corresponding controls. At 24 h after transfection, cells were incubated with 25 μM MG132 for 6 h. Cells were treated with IP lysis buffer, and IP assay was executed with myc tag antibody following the aforementioned procedures. Western blot analysis was applied to measure all the proteins.

Tang Y., Tian W., Zheng S., Zou Y., Xie J., Zhang J., Li X., Sun Y., Lan J., Li N., Xie X, & Tang H. (2023). Dissection of FOXO1-Induced LYPLAL1-DT Impeding Triple-Negative Breast Cancer Progression via Mediating hnRNPK/β-Catenin Complex. Research, 6, 0289.

+ Open protocol

+ Expand

Polysome Profiling of eIF2α Mutant Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were seeded in 15 cm dishes (1 × 10⁵ cells/ml) and cultured overnight (1 dish of HeLa cells; 4 dishes of eIF2α S/S or A/A cells per condition). When indicated, cells were treated with 250 nM of Torin1 (Tocris Bioscience), DMSO or 2.5 μg/ml Tm for 2 hours, prior to polysome analysis. Cells were then incubated 5 minutes with 100 μg/ml of cycloheximide (CHX, Sigma-Aldrich), washed twice with ice-cold PBS containing 100 μg/ml of cycloheximide (PBS + CHX) and harvested in 10 mL of PBS + CHX. Cells were then spun for 5 minutes at 200 g and 4°C and resuspended in 425 μL of hypotonic buffer (5 mM Tris-HCl pH 7.5, 2.5 mM MgCl₂, 1.5 mM KCl, 1 x protease inhibitor cocktail EDTA free (Roche Life Science), 2.5 μL of 20 μg/ml CHX, 1 μL of 1M DTT, 100 U RNase inhibitor (RNasin® Ribonuclease Inhibitors, Promega)). After vortexing the samples for 5 s, 25 μL of 10% Triton X-100 and 25 μL of 10% sodium deoxycholate were added. The samples were then vortexed again for 5 s and centrifuged 7 minutes at 16,000 g and 4°C. 10% of the lysate was kept for determination of cytosolic steady-state mRNA levels and 430 μL were loaded in a 5%–50% sucrose gradient. The gradient was centrifuged at 36,000 g at 4°C for 2 hours and sampled using the Biocomp gradient station (Biocomp) with constant monitoring of optical density at 254 nm.

Schneider K., Nelson G.M., Watson J.L., Morf J., Dalglish M., Luh L.M., Weber A, & Bertolotti A. (2020). Protein Stability Buffers the Cost of Translation Attenuation following eIF2α Phosphorylation. Cell Reports, 32(11), 108154.

+ Open protocol

+ Expand

Polysome Analysis of Transfected Cells

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Polysome analysis was performed with both manual fractionation and RNA analysis^{69 (link)}. Transfected HEK293T cells were incubated with 100 μg/ml cycloheximide (Sigma) for 15 minutes for 37 °C and then washed with ice-cold PBS with 100 μg/ml cycloheximide. Cells were then lysed in polysome buffer, 10 mM Tris pH8, 140 mM NaCl, 1.5 mM MgCl₂, 0.5% v/v NP40, 100 μg/ml cycloheximide, protease inhibitor cocktail (all from Sigma) and 800U/ml RNase OUT (ThermoFisher), for 10 minutes on ice. Cell lysate was centrifuged at 10, 000 × g for 1 minute at 4 °C and supernatant was then adjusted to 200 μg/ml cycloheximide and 700 μg/ml heparin (Sigma). Following centrifugation at 12, 000 × g for 10 minutes at 4 °C, an aliquot of supernatant was taken as input and the rest was layered onto a 10% to 50% sucrose gradient prepared using polysomal buffer. Gradients were then ultracentrifuged at 300,000 × g for 16 hours at 4 °C (Sorvall). After centrifugation, 20 550 μl fractions were collected from the top of the gradients for immunoblotting and quantitative PCR analyses. Level of RNA in fractions was measured by absorbance at 254 nm (Nanodrop) and RNA was precipitated with 5x volume of absolute ethanol (Sigma) overnight at −20 °C.

Lee W.Y., Fu R.M., Liang C, & Sloan R.D. (2018). IFITM proteins inhibit HIV-1 protein synthesis. Scientific Reports, 8, 14551.

+ Open protocol

+ Expand

Investigating WDR5 Protein Stability

Check if the same lab product or an alternative is used in the 5 most similar protocols

After 24 h of culture in a 6-well plate, cycloheximide (CHX, Sigma-Aldrich) was added to each well at a concentration of 50 µg/mL. For the PANC-1 cells transfected with sgVector or sgRNA A1×B2 plasmids, the protein was isolated after 0, 2, 4, 8 or 10 h of CHX treatment. Simultaneously, PANC-1 cells transfected with sgVector or sgRNA A1×B2 plasmids were incubated with MG132 (MedChemExpress) at 10 µM for 10 h. The wild-type PANC-1 cells were treated with 1,6-hexanediol (10 mg/mL, Sigma-Aldrich) for about 2 h, CHX was added for 0, 2 or 4 h, then the protein was harvested and the expression of WDR5 was determined using western blotting assays.

Li W., Wu L., Jia H., Lin Z., Zhong R., Li Y., Jiang C., Liu S., Zhou X, & Zhang E. (2021). The low-complexity domains of the KMT2D protein regulate histone monomethylation transcription to facilitate pancreatic cancer progression. Cellular & Molecular Biology Letters, 26, 45.

+ Open protocol

+ Expand

Protein Stability Regulation in Cell Lines

Check if the same lab product or an alternative is used in the 5 most similar protocols

DNA transfection was performed using Lipofectamine 3000 following the manufacturer’s protocol directly on cells plated on the previous day, in ibidi chambers (for Imaging) or six well plates (for Biochemistry). Experiments were performed 24 h post DNA transfection. siRNA (50 nM final conc) was reverse transfected in six well plates using Lipofectamine RNAiMAX at 0 h and repeated at 24 h. The cells were split at 48 h and either seeded in ibidi chambers (for imaging) or passaged into six well plates (for protein) to be analyzed at a 72 h time point.
Cells were seeded a day before in ibidi chambers and treated with either DMSO, 100 nM thapsigargin (#T7458; Life Technologies) or 50 μM iPDI (PDI inhibitor 16F16, # SML0021; Sigma-Aldrich) in growth media for 1 or 2 h before fixation and IF. For DTT rescue experiment, cells in ibidi chambers were treated with media (growth for MBs/differentiation for MTs) containing either DI water (Vehicle) or 5 μM final conc. of DTT (#AC16568; Acros Organics) for 3 min before fixation and IF.
For CHX chase experiment, the cells were seeded in six-well plates the previous day and treated either with 200 μg/ml CHX (# C-7698; Sigma-Aldrich) in fresh growth media for 2, 4 or 8 h; or without CHX (0 h). The cells were harvested by trypsinization and flash-frozen cell pellets were stored in −20°C for future protein analysis.

Sharma R, & Hetzer M.W. (2023). Disulfide bond in SUN2 regulates dynamic remodeling of LINC complexes at the nuclear envelope. Life Science Alliance, 6(8), e202302031.

+ Open protocol

+ Expand

Brassinosteroid Signaling Regulation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Ten-day-old Col-0, ubp12-2w ubp13-3 and UBP13OE seedlings were transferred to liquid half-strength MS medium with DMSO, 200 nM BL (Wako), 1 μM BRZ (Santa Cruz Biotechnology, Dallas, Texas, USA), 2 μM PCZ (Aladdin), or 50 μM bikinin (Sigma-Aldrich) for 2 h to accumulate different forms of BES1. For experiments using inhibitors, pretreated plants were transferred to liquid half-strength MS medium containing 1 mM CHX (Sigma-Aldrich), 1 mM CHX + 50 μM MG132 (Sigma-Aldrich), 1 mM CHX + 50 μM E64d (Sigma-Aldrich), or 1 mM CHX + 1 μM ConA (Sigma-Aldrich) for indicated times, and then detected the protein changes by immunoblotting using anti-BES1 and anti-ACTIN antibodies. The inhibitor source details are listed in Supplemental Table S2.

Xiong J., Yang F., Yao X., Zhao Y., Wen Y., Lin H., Guo H., Yin Y, & Zhang D. (2022). The deubiquitinating enzymes UBP12 and UBP13 positively regulate recovery after carbon starvation by modulating BES1 stability in Arabidopsis thaliana. The Plant cell, 34(11).

+ Open protocol

+ Expand

Mammalian Oocyte Fertilization and Culture

Check if the same lab product or an alternative is used in the 5 most similar protocols

For DMSO, CHX (Sigma-Aldrich), and AMA (Sigma-Aldrich) treatment, MII oocytes were fertilized with sperms in HTF medium with CHX or AMA (20 μg/ml). Approximately 6 hours after IVF, zygotes with two PN were transferred to KSOM (Millipore) with CHX or AMA (20 μg/ml) and cultured at 37°C under 5% CO₂. DMSO (0.2%, v/v) was used as a control.

Zhang C., Wang M., Li Y, & Zhang Y. (2022). Profiling and functional characterization of maternal mRNA translation during mouse maternal-to-zygotic transition. Science Advances, 8(5), eabj3967.

+ Open protocol

+ Expand

Ribosomal Profiling of Cellular Transcripts

Check if the same lab product or an alternative is used in the 5 most similar protocols

H1299 cells were transduced with shCtrl or shSFTA1P. After 72 h, cells were incubated with CHX (100 mg/mL, Sigma-Aldrich) for 10 min and washed with PBS supplemented with 100 mg/mL CHX, followed by isolation of the cytoplasmic lysates in lysis buffer [10 mM Tris-HCl (pH 7.4), 5 mM MgCl₂, 100 mM KCl, 2 mM DTT, 100 U/ml, SUPERase-In 100 U/ml, 100 μg/ml CHX (C4859, Sigma-Aldrich), Protease inhibitor (1XcOmplete, EDTA-free, Sigma-Aldrich). The crude lysates were then fractionated by ultracentrifugation through 15–50% linear sucrose gradients. In total, 15 fractions were collected and subjected to Phenol–Chloroform extraction followed by glycogen precipitation. RNA extracted from each fraction was analyzed by RT-qPCR.

Zhu B., Finch-Edmondson M., Leong K.W., Zhang X., V. M., Lin Q.X., Lee Y., Ng W.T., Guo H., Wan Y., Sudol M, & DasGupta R. (2021). LncRNA SFTA1P mediates positive feedback regulation of the Hippo-YAP/TAZ signaling pathway in non-small cell lung cancer. Cell Death Discovery, 7, 369.

+ Open protocol

+ Expand

Analyzing Ribosome Profiles in Isogenic p53 Cell Lines

Check if the same lab product or an alternative is used in the 5 most similar protocols

HCT-116 p53^+/+, HCT-116 p53^−/− (kindly provided by Galina Selivanova, Karolinska Institutet) and MCF7 (ATCC HTB-22™) cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 1% l-glutamine (Gibco, Life Technologies). Briefly, cells (1 × 10⁶) were seeded in 15-cm cell culture dishes (Corning), harvested at 80% confluency (HCT-116 p53^+/+ and p53^−/− cells were also serum starved [0.1% fetal bovine serum for 16 h prior to harvest]), lysed in hypotonic lysis buffer (5 mM Tris–HCl, pH 7.5, 2.5 mM MgCl₂, 1.5 mM KCl, 100 μg/ml cycloheximide, 2 mM DTT, 0.5% Triton, 0.5% sodium deoxycholate; all from Sigma Aldrich) and the cytosolic extract was loaded onto the sucrose gradient. A detailed protocol described previously (28 ) was applied with the modification that instead of adding cycloheximide to the media, the cell media was discarded, plates were placed on ice and immediately washed in an ice-cold solution of 1× phosphate-buffered saline (PBS) and cycloheximide (100 μg/ml; Sigma Aldrich).

Liang S., Bellato H.M., Lorent J., Lupinacci F.C., Oertlin C., van Hoef V., Andrade V.P., Roffé M., Masvidal L., Hajj G.N, & Larsson O. (2017). Polysome-profiling in small tissue samples. Nucleic Acids Research, 46(1), e3.

+ Open protocol

+ Expand

Isolation of Translationally Active mRNA

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

This protocol was adapted as previously described²¹ (link) and modified accordingly. In brief, we prepared the cytoplasmic extracts by harvesting proband and control cells in ice-cold PBS containing 100 μg/mL cycloheximide (Sigma). Cells were counted, and 100,000 cells were incubated with 800 μL of RPMI medium containing 10% fetal bovine serum and 100 μg/mL cycloheximide (Sigma) for 5 min at 37°C. After incubation, 200 μL of N-hydroxysuccimide ester (DSP; 1 mM; Pierce) was introduced as a cross-linking reagent and incubated for 5 min at 37°C followed by quenching with 1 M Tris–HCl (pH 7.4). The cells were washed twice by centrifugation at 1,000 r.p.m. for 3 min and rinsed with ice-cold PBS containing 100 μg/mL cycloheximide (Sigma). The final pellets were swollen for 20 min in 500 μL of low-salt buffer (LSB) (20 mM HEPES [pH 7.4], 100 mM KCl, and 2 mM MgCl₂) containing 1 mM dithiothreitol and lysed by the addition of 500 μL lysis buffer (1× LSB containing 1.2% Triton X-100) (Sigma) followed by brief vortexing. One-tenth (70 μL) of the above lysate was transferred to the Ig-coated beads, and incubation was carried out for 2 h at 4°C. After incubation with the HSP70/HSP73 antibody-conjugated magnetic beads, the polysome complexes containing translationally active mRNA transcripts were isolated and eluted from beads with the Array Pure Nanoscale RNA Purification Kit (Epicentre).

Tummala H., Walne A., Buccafusca R., Alnajar J., Szabo A., Robinson P., McConkie-Rosell A., Wilson M., Crowley S., Kinsler V., Ewins A.M., Madapura P.M., Patel M., Pontikos N., Codd V., Vulliamy T, & Dokal I. (2022). Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita. American Journal of Human Genetics, 109(8), 1472-1483.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!