SignalSilence? KLHL12 siRNA II

• 产品编号：CST-9420S      品牌：CST       原厂货号：9420S
• 产品分类：DNA和RNA纯化 > RNA干扰 > RNAi Oligos > siRNA
• 应用分类：

描述：

KLHL12 siRNA II 抑制人和猴中KLHL12的表达。来自Cell Signaling Technology (CST)的SignalSilence^® KLHL12 siRNA II 可以帮助研究者通过RNA干扰特异性地抑制KLHL12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^®KLHL12 siRNA II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。Cullins是以SCF(Skp1-CUL1-F-box)为代表的模式化的泛素连接酶的分子支架蛋白(1-3)。这些E3连接酶的底物选择，主要是通过特定的模块与cullins的结合实现。在SCF复合物中，这一分子由直接与CUL1结合的Skp1，以及F-box蛋白家族诸如Skp2的一个成员组成(1-4)。CUL3已经被证实是哺乳动物和秀丽隐杆线虫胚胎发育所必需(5-7)，但最近，它的底物特异性接合体尚未被阐明。目前已经证实，基于CUL3的泛素连接酶复合物的底物调节剂，包含了一种保守的BTB/POZ (Pox virus and Zinc finger)结构域。这一结构域，最初在果蝇转录抑制复合物中被发现，并存在于190多种人蛋白。BTB蛋白包括许多蛋白-蛋白相互作用结构域，包括MATH结构域，锌指重复序列和kelch重复序列(8)。一系列的证据表明Kelch-like 12 protein (KLHL12)是基于CUL3的泛素连接酶复合物的底物特异调节剂。有关KLHL12氨基酸序列的分析表明，BTB氨基末端的模序，一个中心的链接区域，以及由kelch重复序列组成的羧基末端kelch结构域。此外，已经证实KLHL12，通过散乱的结合，以及为泛素依赖的蛋白酶体降解的靶向作用，可以负向调节Wnt信号转导(9)。此外最近，KLHL12被证实通过促进COPII组分Sec31单泛素化修饰，可以促进大COPII小囊泡的组装。因此，CUL3-KLHL12-依赖的泛素化是胶原排出的必需的，其更是整合素依赖的鼠胚胎干细胞分裂所必需的步骤(10)。

1. Zheng, N. et al. (2002) Nature 416, 703-9.
2. Skowyra, D. et al. (1997) Cell 91, 209-19.
3. Feldman, R.M. et al. (1997) Cell 91, 221-30.
4. Bai, C. et al. (1996) Cell 86, 263-74.
5. Singer, J.D. et al. (1999) Genes Dev 13, 2375-87.
6. Winston, J.T. et al. (1999) Genes Dev 13, 2751-7.
7. Kurz, T. et al. (2002) Science 295, 1294-8.
8. Collins, T. et al. (2001) Mol Cell Biol 21, 3609-15.
9. Angers, S. et al. (2006) Nat Cell Biol 8, 348-57.
10. Jin, L. et al. (2012) Nature 482, 495-500.

原厂资料：

Homology

Species predicted to react based on 100% sequence homology: Monkey

Specificity / Sensitivity

KLHL12 siRNA II will inhibit human and monkey KLHL12 expression.

Description

SignalSilence® KLHL12 siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit KLHL12 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® KLHL12 siRNA II 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

Cullins are proteins that function as molecular scaffolds for modular ubiquitin ligases typified by the SCF (Skp1-CUL1-F-box) complex (1-3). The substrate selectivity of these E3 ligases is dictated by a specificity module that binds cullins. In the SCF complex, this module is composed of Skp1, which binds directly to CUL1, and a member of the F-box family of proteins such as Skp2 (1-4). CUL3 has been shown to be required for embryonic development in mammals and Caenorhabditis elegans (5-7) but until recently, its substrate specificity adaptor had yet to be elucidated. It is now recognized that substrate adaptors for CUL3-based ubiquitin ligase complexes contain a conserved BTB/POZ (Pox virus and Zinc finger) domain. This domain, which was initially identified in the Drosophila transcriptional repressors broad complex, tramtrack, and bric-a-brac is present in more than 190 human proteins. BTB proteins contain a variety of putative protein-protein interaction domains, including MATH domains, zinc finger repeats, and kelch repeats (8). 
 
 There are several lines of evidence suggesting that Kelch-like 12 protein (KLHL12) is a substrate-specific adaptor for the CUL3-based ubiquitin ligase complex. Analysis of the amino acid sequence of KLHL12 reveals an amino-terminal BTB motif, a central linker region, and a carboxy-terminal kelch domain composed of kelch repeats. Furthermore, KLHL12 has been shown to negatively regulate Wnt signaling by binding Disheveled and targeting it for ubiquitin-dependent proteasomal degradation (9). More recently, KLHL12 was shown to drive the assembly of large COPII vesicles by promoting the monoubiquitination of the COPII component Sec31. As a result, CUL3-KLHL12-dependent ubiquitination is essential for collagen export, a step that is required for integrin-dependent mouse embryonic stem cell division (10).

1. Zheng, N. et al. (2002) Nature 416, 703-9.
2. Skowyra, D. et al. (1997) Cell 91, 209-19.
3. Feldman, R.M. et al. (1997) Cell 91, 221-30.
4. Bai, C. et al. (1996) Cell 86, 263-74.
5. Singer, J.D. et al. (1999) Genes Dev 13, 2375-87.
6. Winston, J.T. et al. (1999) Genes Dev 13, 2751-7.
7. Kurz, T. et al. (2002) Science 295, 1294-8.
8. Collins, T. et al. (2001) Mol Cell Biol 21, 3609-15.
9. Angers, S. et al. (2006) Nat Cell Biol 8, 348-57.
10. Jin, L. et al. (2012) Nature 482, 495-500.

注意事项：

Storage: KLHL12 siRNA II is supplied in RNAse-free water.
Aliquot and store at -20ºC.