Chemical structure of cycloheximide.
Western blot analysis of extracts from Jurkat cells, untreated (-) , or treated with Cycloheximide (50 μg/ml, 24 hr; +), Bortezomib #2204 (10 nM, 24 hr; +), or both, using Ubiquitin Antibody #3933 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from Jurkat cells, untreated (-) or treated with increasing concentrations of Cycloheximide (24 hr), using PARP Antibody #9542 (upper), Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb #5625 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Cycloheximide is supplied as a lyophilized powder. For a 10 mg/ml stock, carefully weigh out and reconstitute 50 mg in 5 ml DMSO or EtOH. Working concentrations and length of treatments vary depending on the desired effect, but it is typically used at 5-50 µg/ml for 4-24 hours. Soluble in DMSO, EtOH, or MeOH.
Store lyophilized or in solution at -20ºC, desiccated. Protect from light. In lyophilized form, the chemical is stable for 24 months. Once in solution, use within 3 months to prevent loss of potency. Aliquot to avoid multiple freeze/thaw cycles.
Cycloheximide is a protein synthesis inhibitor in eukaryotes. Although its precise mechanism of action has yet to be fully elucidated, it has been shown to inhibit translation elongation through binding to the E-site of the 60S ribosomal unit and interfering with deacetylated tRNA (1-3). Although not all cell types are equally sensitive to the apoptosis-inducing effects of cycloheximide, it has been shown to induce cell death in T cells through a FADD-dependent mechanism (4). In addition, cycloheximide and Tumor Necrosis Factor possess a synergistic cytotoxicity (5,6), and consequently they are routinely used together to induce cell death. Investigators have demonstrated that cycloheximide blocks bortezomib-stimulated protein ubiquitination (7).
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