SignalSilence? ISG15 siRNA I

• 产品编号：CST-12557S      品牌：CST       原厂货号：12557S
• 产品分类：DNA和RNA纯化 > RNA干扰 > RNAi Oligos > siRNA
• 应用分类：

描述：

来自Cell Signaling Technology (CST)的SignalSilence^® ISG15 siRNA I可以帮助研究者通过RNA干扰特异性地抑制 ISG15的表达，这种方法可以通过将双链RNA分子传递到细胞内从而使基因表达有选择的沉默。来自CST的所有的SignalSilence^® siRNA产品都是经过内部严格检测的，并且通过Western blot 分析证明确实能够减少目的蛋白的表达。通过三苯甲基分析每个碱基以监测寡核苷酸的合成，确保合适的配对效率。随后寡核苷酸通过亲和固相萃取法纯化。退火的RNA双链通过质谱分析来证实其精确的组成。每一批产品都通过质谱分析与前面的产品进行比较，来保证不同批次之间的最大一致性。CST推荐使用100 nM SignalSilence^®ISG15 siRNA I进行转染，48到72小时后对细胞进行裂解。转染步骤按照转染试剂说明书提供的步骤进行。遇到任何使用方面的问题，请随时联系CST。每小瓶可供100次转染，每次转染量相当于在转染24孔板时，每孔总体积为300μl培养基中siRNA的终浓度为100nM。ISG15（Interferon-stimulated 15 kDa protein）是的15 kDa的干扰素刺激蛋白，也称为泛素反应性蛋白(UCRP)，是泛素样蛋白家族的成员之一，并在从妊娠到天然免疫反应等多种生物学过程中发挥作用(1)。ISG15的表达由暴露于细胞的I型干扰素α和β刺激，除外病毒感染及流感B (2,3)。暴露于I型干扰素后，除外一些成纤维细胞核上皮细胞，淋巴细胞和单核细胞释放ISG15到培养基(1,4)。ISG15已经被证实作为细胞因子，通过单核细胞和巨噬细胞，刺激干扰素γ的分泌、自然杀伤细胞的增殖和中性粒细胞的趋化效应(4,5)。ISG15也被证实在细胞内发挥作用，通过E1 (Ube1L), E2 (UbcH8) 和E3连接酶通过多种类似于泛素化作用的步骤与其他蛋白共价结合(6,7)。ISG15通过泛素化加工蛋白酶Ubp43，与其它蛋白解离(8)。ISG15-蛋白结合物(ISGylation)由I型干扰素诱导，并作用于包括丝氨酸蛋白抑制Serpin 2A, PLCγ1, ERK1/2, Jak1和Stat1等靶向蛋白(9,10)。不同于泛素化作用，ISGylation并不介导蛋白的降解，相反ISGylation提高了Jak1和Stat1的活性，加强了对干扰素的细胞应答(11)。

1. Ritchie, K.J. and Zhang, D.E. (2004) Semin. Cell Dev. Biol. 15, 237-246.
2. Korant, B.D. et al. (1984) J. Biol. Chem. 259, 14835-14839.
3. Haas, A.L. et al. (1987) J. Biol. Chem. 262, 11315-11323.
4. Knight, E. and Cordova, B. (1991) J. Immunol. 146, 2280-2284.
5. D'Cunha, J. et al. (1996) Proc. Natl. Acad. Sci. USA 93, 211-215.
6. Loeb, K.R. and Haas, A.L. (1992) J. Biol. Chem. 267, 7806-7813.
7. Zhao, C. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 10200-10205.
8. Malakhov, M.P. et al. (2002) J. Biol. Chem. 277, 9976-9981.
9. Malakhov, M.P. et al. (2003) J. Biol. Chem. 278, 16608-16613.
10. Hamerman, J.A. et al. (2002) J. Immunol. 168, 2415-2423.
11. Malakhova, O.A. et al. (2003) Genes Dev. 17, 455-460.

原厂资料：

Description

SignalSilence® ISG15 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit ISG15 expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.

Quality Control

Oligonucleotide synthesis is monitored base by base through trityl analysis to ensure appropriate coupling efficiency. The oligo is subsequently purified by affinity-solid phase extraction. The annealed RNA duplex is further analyzed by mass spectrometry to verify the exact composition of the duplex. Each lot is compared to the previous lot by mass spectrometry to ensure maximum lot-to-lot consistency.

Directions for Use

CST recommends transfection with 100 nM SignalSilence® ISG15 siRNA I 48 to 72 hours prior to cell lysis. For transfection procedure, follow protocol provided by the transfection reagent manufacturer. Please feel free to contact CST with any questions on use. 
 Each vial contains the equivalent of 100 transfections, which corresponds to a final siRNA concentration of 100 nM per transfection in a 24-well plate with a total volume of 300 μl per well.

Background

Interferon-stimulated 15 kDa protein (ISG15), also known as ubiquitin cross-reactive protein (UCRP), is a member of the ubiquitin-like protein family and functions in various biological pathways from pregnancy to innate immune responses (1). Expression of ISG15 is stimulated by cellular exposure to type 1 interferons α and β, in addition to infection with viruses such as influenza B (2,3). After exposure to type I interferons, both lymphocytes and monocytes, in addition to some fibroblasts and epithelial cells, release ISG15 into culture medium (1,4). ISG15 has been shown to function as a cytokine, stimulating interferon γ secretion by monocytes and macrophages, proliferation of natural killer cells, and chemotactic responses in neutrophils (4,5). ISG15 has also been shown to function intracellularly, being covalently conjugated to other proteins by E1 (Ube1L), E2 (UbcH8) and E3 ligases via a multi-step process analogous to ubiquitination (6,7). ISG15 is removed from proteins by the ubiquitin processing protease Ubp43 (8). ISG15-protein conjugation (ISGylation) is induced by type 1 interferons, and target proteins include the serine protease inhibitor Serpin 2A, PLCγ1, ERK1/2, Jak1 and Stat1 (9,10). Unlike ubiquitination, ISGylation does not target proteins for degradation, rather ISGylation increases Jak1 and Stat1 activity, enhancing the cellular response to interferons (11).

1. Ritchie, K.J. and Zhang, D.E. (2004) Semin. Cell Dev. Biol. 15, 237-246.
2. Korant, B.D. et al. (1984) J. Biol. Chem. 259, 14835-14839.
3. Haas, A.L. et al. (1987) J. Biol. Chem. 262, 11315-11323.
4. Knight, E. and Cordova, B. (1991) J. Immunol. 146, 2280-2284.
5. D'Cunha, J. et al. (1996) Proc. Natl. Acad. Sci. USA 93, 211-215.
6. Loeb, K.R. and Haas, A.L. (1992) J. Biol. Chem. 267, 7806-7813.
7. Zhao, C. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 10200-10205.
8. Malakhov, M.P. et al. (2002) J. Biol. Chem. 277, 9976-9981.
9. Malakhov, M.P. et al. (2003) J. Biol. Chem. 278, 16608-16613.
10. Hamerman, J.A. et al. (2002) J. Immunol. 168, 2415-2423.
11. Malakhova, O.A. et al. (2003) Genes Dev. 17, 455-460.

注意事项：

Storage: ISG15 siRNA I is supplied in RNAse-free water. Aliquot
and store at -20ºC.