SignalSilence? Atg4A siRNA I

• 产品编号：CST-6427S      品牌：CST       原厂货号：6427S
• 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^® Atg4A siRNA I 可以帮助研究者通过RNA干扰特异性地抑制 Atg4A 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。自噬是自噬溶酶体对其包裹的细胞质内容物进行降解的一种分解代谢过程(1)。关于自噬的调控的研究，大部分集中在酵母中，还包含一些由自噬相关基因(Atg)编码的蛋白。自噬小泡的形成需要一对重要的泛素样连接体系，Atg12-Atg5和Atg8-磷脂酰乙醇胺（Atg8-PE），这些广泛存在于真核细胞中(2)。与酵母相对应的许多哺乳动物的Atg蛋白已被描述，包括三种Atg8蛋白(GATE-16、GABARAP, 和LC3 )和四种Atg4同源物（(Atg4A/自噬蛋白酶-2、Atg4B/自噬蛋白酶-1、Atg4C/自噬蛋白酶-3 和 Atg4D/自噬蛋白酶-4)(3-5)。半胱氨酸蛋白酶Atg4对自噬体膜的生成和调控至关重要。Atg4通过剪切Atg8的羰基末端，并暴露其E1样酶Atg7甘氨酸残基，起始Atg8同源物的脂化。Atg8同源物转移到E2样酶Atg3，之后形成Atg8-PE结合物。在自噬的晚期，Atg4可以通过剪切PE逆转这种脂化，从而逆转Atg8同源物(6)。

1. Reggiori, F. and Klionsky, D.J. (2002) Eukaryot Cell 1, 11-21.
2. Ohsumi, Y. (2001) Nat Rev Mol Cell Biol 2, 211-6.
3. Kabeya, Y. et al. (2000) EMBO J 19, 5720-8.
4. Kabeya, Y. et al. (2004) J Cell Sci 117, 2805-12.
5. Mariño, G. et al. (2003) J Biol Chem 278, 3671-8.
6. Sou, Y.S. et al. (2008) Mol Biol Cell 19, 4762-75.

原厂资料：

Description

SignalSilence® Atg4A siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Atg4A 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 Atg4A 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.

Background

Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents. Control of autophagy was largely discovered in yeast and involves proteins encoded by a set of autophagy-related genes (Atg) (1). Formation of autophagic vesicles requires a pair of essential ubiquitin-like conjugation systems, Atg12-Atg5 and Atg8-phosphatidylethanolamine (Atg8-PE), which are widely conserved in eukaryotes (2). Numerous mammalian counterparts to yeast Atg proteins have been described, including three Atg8 proteins (GATE-16, GABARAP, and LC3) and four Atg4 homologues (Atg4A/autophagin-2, Atg4B/autophagin-1, Atg4C/autophagin-3, and Atg4D/autophagin-4) (3-5). The cysteine protease Atg4 is pivotal to autophagosome membrane generation and regulation. Atg4 primes the Atg8 homologue for lipidation by cleaving its carboxy terminus and exposing its glycine residue for E1-like enzyme Atg7. The Atg8 homologue is transferred to the E2-like enzyme Atg3 before forming the Atg8-PE conjugate. During later stages of autophagy, Atg4 can reverse this lipidation event by cleaving PE, thereby recycling the Atg8 homologue (6).

1. Reggiori, F. and Klionsky, D.J. (2002) Eukaryot Cell 1, 11-21.
2. Ohsumi, Y. (2001) Nat Rev Mol Cell Biol 2, 211-6.
3. Kabeya, Y. et al. (2000) EMBO J 19, 5720-8.
4. Kabeya, Y. et al. (2004) J Cell Sci 117, 2805-12.
5. Mariño, G. et al. (2003) J Biol Chem 278, 3671-8.
6. Sou, Y.S. et al. (2008) Mol Biol Cell 19, 4762-75.

注意事项：

Storage: Atg4A siRNA I is supplied in RNAse-free water. Aliquot
and store at -20ºC.