| 名稱 | Ro 31-8220 Mesylate |
| 描述 | Ro 31-8220 Mesylate (Bisindolylmaleimide IX mesylate) is a pan-PKC inhibitor for PKC-α/βI/βII/γ/ε (IC50: 5/24/14/27/24 nM), and also shows potent inhibition against MSK1, MAPKAP-K1b, S6K1, and GSK3β. |
| 細胞實驗 | Human A549 lung and MCF-7 breast carcinoma cells are obtained from the European Collection of Animal Cell Cultures. Cells (passage number 10-30) are cultured in an atmosphere of 5% carbon dioxide, the former in Ham's F-12 medium with penicillin/streptomycin, the latter in minimum essential medium (Eagle's modification) with additional pyruvate (1 mM) and non-essential amino acids. Both media are supplemented with 10% FCS and glutamine (2 mM). Cells are subcultured routinely twice weekly to maintain logarithmic growth. For cell proliferation studies cells are seeded and incubated with 3 ml of medium including agents, which is replenished at intervals of 48 h (A549) or 72 h (MCF-7). Following incubation for 4 days (A549) or 6 days (MCF-7) with drugs, cell number is assessed using a Coulter Counter Model ZM. In order to achieve PKC depletion, cells are incubated for 24 h with bryostatin 1 (1 μM). Under these conditions growth inhibition caused by bryostatin 1 is negligible. Bryostatin is removed by extensive washing of the cells followed by a 2 h recovery period. In previous work using the A549 cell line this washing procedure has been shown to eliminate bryostatin-mediated effects. The cells are then incubated for a further 24 h with staurosporine, RO 31-8220, UCN-01 or H-7. In some experiments cells are incubated with inhibitor for 48 rather than 24 h, in this case bryostation was not removed and left in the incubate. After removal of agents inhibition of DNA synthesis is evaluated by measurement of [3H]Tdr incorporation into cell. Radioactivity is counted using a Packard 1500 Tricarb scintillation counter. (Only for Reference) |
| 激酶實驗 | Assay of PKC Activity : Assay mixtures contain 0.2 mg/mL peptide-gamma, 10 μM MgCl2, 0.6 mM CaCl2, 10 μM [γ-32P]ATP, 1.25 mg/mL phosphatidylserine and 1.25 ng/mL phorbol 12-myristate 13-acetate in 20 mM Hepes (pH 7.5), 2 mM EDTA, 1 mM dithiothreitol and 0.02% (w/v) Triton X-100. Peptide-γ is a synthetic peptide, GPRPLFCRKGSLRQKW, resembling the PKC-γ pseudosubstrate site, except that a serine residue replaces the pseudosubstrate alanine, converting the peptide from an inhibitor into a substrat. The assays are started by the addition of 2.5 m-units of enzyme, incubated at 30 °C for 10 min and terminated by spotting on to P81 paper, followed by extensive washing in 75 mM orthophosphoric acid. The papers are then washed in ethanol, dried, and incorporated radioactivity is determined by liquidscintillation spectroscopy. |
| 體外活性 | 在MLP?/?小鼠體內(nèi),Ro 31-8220 (6 mg/kg/d,s.c.)可明顯增加心肌收縮力. |
| 體內(nèi)活性 | RO31-8220可有效抑制A549細胞（IC50：0.78 μM）和MCF-7細胞（IC50：0.897 μM）的生長。在兒茶酚胺低反應(yīng)血小板中,RO31-8220可增強Akt的磷酸化從而使腎上腺素誘導的血小板聚集作用增強。通過抑制apoE基因的囊泡運輸?shù)劫|(zhì)膜,RO31-8220對載脂蛋白E從原代人巨噬細胞的分泌有顯著降低效果,對ApoE的mRNA水平或蛋白水平無顯著影響。RO31-8220抑制大鼠腦蛋白激酶C活性（IC50：23 nM）,對PKC-α/β/γ/ε無選擇性。 此外,RO31-8220對電壓依賴性鈉離子通道有抑制作用。 |
| 存儲條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice. |
| 溶解度 | Ethanol : 2.8 mg/mL (5 mM)
DMSO : 55.4 mg/mL (100 mM)
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| 關(guān)鍵字 | inhibit | Inhibitor | Ro 318220 Mesylate | Protein kinase C | PKC | Ro 31 8220 Mesylate | Ro 31-8220 Mesylate | Bisindolylmaleimide IX Mesylate | Ro 31-8220 |
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