Western blot analysis of extracts from 293T cells, treated with Anisomycin (25 μg/ml) for the indicated times, with or without pretreatment with JNK inhibitor SP600125 (50 μM, 40 min; +), using Phospho-JunB (Thr102/Thr104) (D3C6) Rabbit mAb #8053 (upper) or JunB (C37F9) Rabbit mAb #3753 (lower).
Western blot analysis of extracts from Jurkat cells, serum-starved overnight, pretreated with the indicated concentrations of SB202190 for 1 hr, and subsequently treated with Anisomycin (25 μg/ml, 30 min; +), using Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb #4511 (upper) or p38 MAPK Antibody #9212 (lower).
Chemical structure of anisomycin.
Anisomycin is supplied as a lyophilized powder. For a 25 mg/ml stock, reconstitute the 10 mg in 400 µl DMSO. Working concentrations and length of treatments vary depending on the desired effect, but it is typically used at 5-50 µg/ml for 5-60 minutes. Soluble in DMSO or MeOH.
Store lyophilized or in solution at 4ºC, 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.
Anisomycin, an antibiotic produced by Streptomyces griseolus and S. roseochromogenes, was originally described to inhibit protein-protein synthesis at the translational level (1). More recently, it is has been well characterized to strongly activate the stress kinases SAPK/JNK and p38 MAPK, as well as p70/85 S6 kinase in mammalian cells, which results in the rapid induction of immediate-early (IE) genes, such as c-fos, fosB, c-jun, JunB, and JunD (1). Investigators have demonstrated that anisomycin acts as a potent signaling agonist, synergizing with growth factors and phorbol esters to superinduce these IE genes (1,2). Research studies have demonstrated that anisomycin induces apoptosis in many cancer cell lines (3-5).
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