Cell Signaling Technology

Product Pathways - Autophagy Signaling

SignalSilence® Atg13 siRNA I #12043

No. Size Price
12043S 300 µl ( 3 nmol ) ¥3,224.00 现货查询 购买询价
12043 carrier free & custom formulation / quantityemail request
Applications Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
TFN Human,

Species cross-reactivity is determined by western blot.

Applications Key: TFN=Transfection,

Homology

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

Specificity / Sensitivity

SignalSilence® Atg13 siRNA I inhibits human, mouse, and monkey Atg13 expression.

SignalSilence® Atg13 siRNA I 能够抑制人、小鼠以及猴源的Atg13的表达。

Description

SignalSilence® Atg13 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Atg13 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.

来自Cell Signaling Technology (CST)的SignalSilence® Atg13 siRNA I 可以帮助研究者通过RNA干扰特异性地抑制Atg13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。

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.

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。

Western Blotting

Western Blotting

Western blot analysis of extracts from RD cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), or SignalSilence® Atg13 siRNA I (+), using Atg13 Antibody #6940 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The Atg13 Antibody confirms silencing of Atg13 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.

Western blot 分析RD细胞的细胞提取物,使用100 nM SignalSilence® Control siRNA (Unconjugated) #6568(-)或SignalSilence® Atg13 siRNA I (+)转染,使用抗体是Atg13 Antibody #6940 (上图)或β-Actin (D6A8) Rabbit mAb 兔单抗#8457 (下图)。Atg13 Antibody 确认Atg13表达的沉默,β-Actin (D6A8) Rabbit mAb 兔单抗主要用于检测内参。

Directions for Use

CST recommends transfection with 100 nM SignalSilence® Atg13 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.

CST推荐使用100 nM SignalSilence® Atg13 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。

Background

Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents (1,2). Autophagy is generally activated by conditions of nutrient deprivation, but has also been associated with a number of physiological processes including development, differentiation, neurodegeneration, infection, and cancer (3). The molecular machinery of autophagy was largely discovered in yeast and referred to as autophagy-related (Atg) genes. 
 
 Atg13/Apg13 was originally identified in yeast as a constitutively expressed protein that was genetically linked to Atg1/Apg1, a protein kinase required for autophagy (4). Over-expression of Atg1 suppresses the defects in autophagy observed in Atg13 mutants (4). Autophagy requires a direct association between Atg1 and Atg13, and is inhibited by TOR-dependent phosphorylation of Atg13 under high nutrient conditions (5). Similarly, mammalian Atg13 forms a complex with the Atg1 homologues ULK1/2, along with FIP200, localizes to autophagic isolation membranes, and regulates autophagosome biogenesis (6-8). mTOR phosphorylates both Atg13 and ULK1, suppressing ULK1 kinase activity and autophagy (7-9). ULK1 can directly phosphorylate Atg13 at a yet unidentified site, presumably to promote autophagy (7,8). Additional studies suggest that Atg13 and FIP200 can function independently of ULK1 and ULK2 to induce autophagy through an unknown mechanism (10).

自噬是自噬溶酶体对其包裹的细胞质内含物进行降解的一种分解代谢过程(1,2)。自噬过程一般是在营养匮乏的条件下被激活,但是也与一些生理过程有关,如发育、分化、神经变性、感染和癌症(3)。在酵母中已阐明自噬的分子机制,这是由许多自噬相关基因(Atg)调控。

Atg13/Apg13作为一种固有蛋白,最早在酵母中被发现,其与自噬必须的蛋白激酶Atg1/Apg1存在遗传上的关联性。从Atg13的突变株发现,Atg1的过表达可以抑制自噬缺陷(4)。自噬的发生需要Atg1和Atg13的直接关联,并且通过TOR依赖的磷酸化Atg1的同系物ULK1/2和FIP2000产生抑制,这种蛋白定位于自噬隔离膜上,并且可以调节自噬体的形成(6-8)。mTOR可以将Atg13和ULK1磷酸化,从而抑制ULK1激酶活性和自噬(7-9)。ULK1可以将Atg13的一个未知的位点磷酸化,从而可能促进自噬(7,8)。另一些研究表明,Atg13和FIP200可以不依赖于ULK1和ULK2诱导自噬,但是机理尚不清楚(10)。

  1. Reggiori, F. and Klionsky, D.J. (2002) Eukaryot Cell 1, 11-21.
  2. Codogno, P. and Meijer, A.J. (2005) Cell Death Differ 12 Suppl 2, 1509-18.
  3. Levine, B. and Yuan, J. (2005) J Clin Invest 115, 2679-88.
  4. Funakoshi, T. et al. (1997) Gene 192, 207-13.
  5. Kamada, Y. et al. (2000) J Cell Biol 150, 1507-13.
  6. Ganley, I.G. et al. (2009) J Biol Chem 284, 12297-305.
  7. Hosokawa, N. et al. (2009) Mol Biol Cell 20, 1981-91.
  8. Jung, C.H. et al. (2009) Mol Biol Cell 20, 1992-2003.
  9. Kim, J. et al. (2011) Nat Cell Biol 13, 132-41.
  10. Alers, S. et al. (2011) Autophagy 7, 1423-33.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

SignalSilence is a registered trademark of Cell Signaling Technology, Inc.

Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.

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