Cell Signaling Technology

Product Pathways - PI3K / Akt Signaling

PathScan® Akt Signaling Antibody Array Kit (Fluorescent Readout) #9700

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No. Size Price
9700S 1 Kit ( 32 multiplexed assays ) ¥8,408.00 现货查询 购买询价
Kit Includes Quantity Applications Reactivity Homology† MW (kDa) Isotype
Detection Antibody Cocktail (10X) 300 µl
Array Slides - Akt Signaling Array 2 ea
16-Well Gasket 2 ea
PathScan® Sandwich ELISA Lysis Buffer (1X) #7018 30 ml
20X Array Wash Buffer 15 ml
DyLight 680TM-linked Streptavidin (10X) 300 µl
Sealing Tape 2 sheets
Array Blocking Buffer 5 ml
Array Diluent Buffer 15 ml

Specificity / Sensitivity

PathScan® Akt Signaling Antibody Array Kit (Fluorescent Readout) detects the indicated cellular proteins and signaling nodes only when phosphorylated on the specified residues (see Array Target Map). No substantial cross-reactivity has been observed between targets. This kit is optimized for cell lysates diluted to a total protein concentration between 0.2 and 1 mg/ml (see kit protocol). All capture antibodies have been validated for human and mouse-derived samples.

PathScan® Akt Signaling Antibody Array试剂盒(Chemiluminescent Readout)能够检测特定位点磷酸化的(见Array靶图)细胞蛋白和信号。不同靶点没有交叉反应。该试剂盒已经针对梯度稀释后的0.2到1mg/ml的总蛋白裂解液进行了优化(见试剂盒说明书)。所有抗体都经过了人和小鼠来源样品的验证。

Description

The PathScan® Akt Signaling Antibody Array Kit (Fluorescent Readout) uses glass slides as the planar surface and is based upon the sandwich immunoassay principle. The array kit allows for the simultaneous detection of 16 phosphorylated proteins predominantly belonging to the Akt signaling network. Target-specific capture antibodies have been spotted in duplicate onto nitrocellulose-coated glass slides. Each kit contains two 16-pad slides, allowing the user to test up to 32 samples and generate 512 data points in a single experiment. Cell lysate is incubated on the slide followed by a biotinylated detection antibody cocktail. Streptavidin-conjugated DyLight® 680 is then used to visualize the bound detection antibody. A fluorescent image of the slide can then be captured with a digital imaging system and spot intensities quantified using array analysis software.

PathScan® Akt Signaling Antibody Array试剂盒(Chemiluminescent Readout)使用玻片作为二维平面,基于三明治免疫分析原理。该试剂盒可以同时检测16种属于Akt信号通路的磷酸化的蛋白。靶点特异性捕获抗体以重复方式定点到硝化纤维覆盖的玻片上。每个试剂盒有2张16-pad玻片,允许使用者检测单次试验最多32个样品,并产生512个数据点。细胞裂解液覆盖到玻片上随后使用生物素标记的抗体混合液进行孵育。链霉亲和素偶联的HRP和LumiGLO®试剂随后用以化学显色抗体。玻片的影像可以用数字显色系统或经典的化学发光膜显色。影像可以被分析或显色强度可以通过array分析软件定量。

Figure 1. Target map of the PathScan® Akt Signaling Antibody Array Kit (Fluorescent Readout) #9700.

Figure 2. MCF7 cells were grown to 85% confluency and then serum starved overnight. Cells were either untreated or treated with Human Insulin-like Growth Factor I (hIGF-I) #8917 (100 ng/ml, 20 min). Cell extracts were prepared and analyzed using the PathScan® Akt Signaling Antibody Array Kit (Fluorescent Readout) #9700. Panel A shows images that were acquired using the LI-COR® Biosciences Odyssey® imaging system. Panel B shows quantification of results. Pixel intensity was quantified using the LI-COR® Image Studio v2.0 array analysis software.

图2.MCF7细胞生长至85%接触抑制,随后血清饥饿过夜。细胞不经过处理(左)或经过人Insulin-like Growth Factor I (hIGF-I) #8917 (100 ng/ml, 20 min; 右)。细胞提取物随后制备好使用PathScan® Akt Signaling Antibody Array试剂盒 (Chemiluminescent Readout) #9700进行分析。Panel A显示了被LI-COR® Biosciences Odyssey® 图像系统捕获的图像。Panel B显示了定量结果。像素强度使用LI-COR® Image Studio v2.0 array分析软件进行定量。

Figure 3. A-431 cells were grown to 85% confluency and then serum starved overnight. Cells were either untreated or treated with Human Epidermal Growth Factor (hEGF) #8916 (100 ng/ml, 5 min). Cell extracts were prepared and analyzed using the PathScan® Akt Signaling Antibody Array Kit (Fluorescent Readout) #9700. Panel A shows images that were acquired using the LI-COR® Biosciences Odyssey® imaging system. Panel B shows quantification of results. Pixel intensity was quantified using the LI-COR® Image Studio v2.0 array analysis software.

图3. A-431细胞生长至85%接触抑制,随后血清饥饿过夜。细胞不经过处理(左)或经过人Epidermal Growth Factor (hEGF) #8916 (100 ng/ml,5 min; 右)。细胞提取物随后制备好使用PathScan® Akt Signaling Antibody Array试剂盒 (Chemiluminescent Readout) #9700进行分析。Panel A显示了被LI-COR® Biosciences Odyssey® 图像系统捕获的图像。Panel B显示了定量结果。像素强度使用LI-COR® Image Studio v2.0 array分析软件进行定量。

Background

The Akt signaling module is typically activated in response to growth factor stimulation of receptor tyrosine kinases transmitting primarily anabolic growth and survival signals. Akt1/2 are ubiquitously expressed protein kinases having a multitude of cellular substrates and are involved in the regulation of a wide range of cellular processes. Akt is activated by phosphorylation at two distinct sites: Ser473 by the mTORC2 complex and Thr308 by the plasma membrane residing kinase PDK1. 
 
 PI3 kinase is a lipid kinase that phosphorylates inositol phospholipids at position three to generate docking sites for Akt at the plasma membrane where Akt is activated. PTEN is a lipid phosphatase that negates the action of PI3 kinase to downregulate the signal emanating from this module. 
 
 mTOR integrates growth factor signaling and nutrient availability and is a core component of two macromolecular complexes, mTORC1 and mTORC2. The autophosphorylation of mTOR at Ser2481 correlates with the levels of its activation. mTORC1 phosphorylation of p70 S6 kinase leads to kinase activation, which in turn activates protein synthesis. The S6 ribosomal protein is found downstream of p70 S6 kinase and its phoshporylation at Ser235/236 reflects mTOR pathway activation. The mTORC2 complex activates Akt by phosphorylating it at Ser473. Phosphorylation of PRAS40 at Thr246 by Akt relieves PRAS40 inhibition of mTORC1. 
 
 4E-BP1 is a repressor of translation and inhibits cap-dependent translation initiation. Hyperphosphorylation of 4E-BP1 by mTORC1 leads to derepression of this blockade, which results in activation of cap-dependent translation. 
 
 Phosphorylation of the pro-apoptotic protein Bad at Ser112 and the multifunctional kinases GSK-3α and GSK-3β at Ser21 and Ser9, respectively, by Akt inhibits their activity and promotes cell survival. 
 
 AMPK is an energy sensor that is activated by phosphorylation at Thr172 in response to elevated AMP levels. Under conditions of low energy and elevated levels of AMP, AMPK helps to ensure that anabolic processes, such as those triggered by Akt, are decreased until energy levels are restored. 
 
 Although not a component of the Akt signaling network, Erk1 and Erk2 kinases are a central component of the Ras/MAP kinase signaling module. Erk1/2 regulate multiple cellular functions and are involved in a broad range of cellular processes, such as proliferation, differentiation, and motility. Erk and Akt signaling modules cross regulate each other at multiple points and through a variety of mechanisms. Erk is activated by a wide range of extracellular signals including growth factors, cytokines, hormones, and neurotransmitters, leading to dual phosphorylation at Thr202 and Tyr204. 
 
 The 90 kDa ribosomal S6 kinase 1 (RSK1) is activated primarily by Erk1/2 in response to many growth factors, polypeptide hormones, and neurotransmitters. p90RSK1 phosphorylates a wide range of substrates including ribosomal protein S6, and positively regulates protein translation and cellular growth. p90RSK1 can also be activated by kinases that regulate the response to cellular stress.

受体酪氨酸激酶转移的类固醇生长和存活信号刺激了生长因子能够激活Akt信号通路。Akt1/2是广泛表达的蛋白激酶有多种细胞底物,涉及到了对多个细胞反应的调控。Akt可以在两个不同的位点被磷酸化:被mTORC2在Ser473磷酸化和被胞膜激酶PDK1磷酸化Thr308. PI3K激酶是一个脂类激酶磷酸化肌醇磷脂的三个位点以形成接纳细胞膜Akt的结构。PTEN是一个脂类磷酸酶能够抑制PI3激酶的活性以下调信号传出。mTOR整合了生长因子信号和营养信号,是两个大分子复合物的重要成分,mTORC1和mTORC2。mTOR的Ser2481的自磷酸化与它的激活相关。
p70 S6磷酸化mTORC1会导致激酶活化,随后激活蛋白合成。S6核糖体蛋白被认为是p70 S6激酶的下游,它Ser235/236的磷酸化表明了mTOR通路的活化。mTORC2复合物通过磷酸化Ser473激活Akt。Akt磷酸化PRAS40的Thr246会释放PRAS40对mTORC1的抑制作用。4E-BP1是翻译的抑制因子,能够抑制cap-依赖的翻译起始。4E-BP1被mTORC1高度磷酸化会去除这种抑制作用,引发cap-依赖的翻译激活。
Akt磷酸化促凋亡蛋白Bad的Ser112以及分别磷酸化多功能激酶GSK-3α和
GSK-3β的Ser21和Ser9,可以抑制它们的功能以及促进细胞生存。AMPK是一个能量传感器,AMP水平上升后磷酸化Thr172能够激活其功能。在低能量,高AMP水平的条件下,AMPK可以帮助确认类固醇突进,例如被Akt引发的信号,AMPK会降低水平指导能量水平恢复。
尽管不是Akt信号通路的组分,Erk1和Erk2激酶是Ras/MAP激酶信号通路的重要成分。Erk1/2 调控多种细胞功能,涉及了广泛的细胞过程,例如增殖,分化和运动。Erk和Akt信号在多个位点通过不同的机制互相调控。Erk能够被多种细胞外信号激活,包括生长因子,细胞因子,激素和神经递质,会导致Thr202和Tyr204的双重磷酸化。生长因子,多肽激素和神经递质刺激后Erk1/2可以激活90kDa核糖体S6激酶1(RSK1)。p90RSK1能够磷酸化多种底物,包括核糖体打那笔S6,并能够正向调控蛋白翻译和细胞生长。p90RSK1也可以被激酶激活以调控细胞压力应激。

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DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.

LI-COR is a registered trademark of LI-COR, Inc.

Odyssey is a registered trademark of LI-COR, Inc.

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

PathScan is a trademark of Cell Signaling Technology, Inc.

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

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