Cell Signaling Technology

Product Pathways - PI3K / Akt Signaling

Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific) #9018

sc-135650  

No. Size Price
9018S 100 µl ( 10 western blots ) ¥4,050.00 现货查询 购买询价
9018T 20 µl ( 2 western blots ) ¥1,600.00 现货查询 购买询价
9018 carrier free & custom formulation / quantityemail request
Applications Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
W 1:1000 Human,Mouse,Rat, Endogenous 60 Rabbit IgG
IP 1:50
F 1:400
IF-IC 1:400

Species cross-reactivity is determined by western blot.

Applications Key: W=Western Blotting, IP=Immunoprecipitation, F=Flow Cytometry, IF-IC=Immunofluorescence (Immunocytochemistry),

Specificity / Sensitivity

Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific) recognizes endogenous levels of Akt1 protein only when phosphorylated at Ser473. It does not detect Akt2 protein when phosphorylated at Ser474.

Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific)兔单抗只识别内源性水平的,在Ser473位点被磷酸化的Akt1,不识别在Ser473位点被磷酸化的Akt2.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser473 of human Akt1 protein.

单克隆抗体由合成重组蛋白免疫动物产生,该重组蛋白与人ALPP蛋白Ser473邻近序列对应。

IF-IC

IF-IC

Confocal immunofluorescent analysis of Akt1 (-/-) mouse embryonic fibroblast (MEF) (left column), or Akt2 (-/-) MEF (right column) cells, stimulated with hPDGF #8913 (100 ng/ml, 15 min) (top row) or inhibited with LY294002 #9901 (bottom row), using Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific) (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Akt1 (-/-)小鼠胚胎成纤维细胞(MEF)(左)或Akt2 (-/-) MEF(右),经过hPDGF #8913 (100 ng/ml, 15 min)(上排)处理或使用LY294002 #9901(下排)处理后,使用Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific)兔单抗进行激光共聚焦免疫荧光分析(绿色)。肌动蛋白丝使用DY-554鬼笔环肽标记(红色)。蓝色伪彩= DRAQ5® #4084 (fluorescent DNA dye).

Western Blotting

Western Blotting

Western blot analysis of extracts from Akt1 (-/-) mouse embryonic fibroblast (MEF) or Akt2 (-/-) MEF, untreated or stimulated with hPDGF #8913 (100 ng/ml, 15 min), using Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific) (upper), Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (middle), or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

Akt1 (-/-)小鼠胚胎成纤维细胞(MEF)或Akt2 (-/-) MEF,不处理或使用hPDGF #8913 (100 ng/ml, 15 min)处理后的细胞提取物,使用Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific)(上),Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb#4060(中),或Akt (pan) (C67E7) Rabbit mAb#4691(下)进行western blot分析。

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of Akt1 (-/-) mouse embryonic fibroblast (MEF) cells (blue) or Akt2 (-/-) MEF cells (green), PDGF-treated (100 ng/mL, 15 min), using Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary Ab.

Akt1 (-/-)小鼠胚胎成纤维细胞(MEF)(蓝色)或Akt2 (-/-) MEF 细胞(绿色),用PDGF处理(100 ng/mL, 15 min),使用Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific)进行流式分析。Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412用作二抗。

Western Blotting

Western Blotting

Western blot analysis of extracts from LNCaP cells, transfected with a construct expressing Akt1 shRNA or Akt2 shRNA, uninduced (-) or doxycycline-induced for the indicated times, using Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific) (top), Phospho-Akt2 (Ser474) (D3H2) Rabbit mAb (Akt2 Specific) #8599 (2nd from top), Akt1 (C73H10) Rabbit mAb #2938 (middle), Akt2 (D6G4) Rabbit mAb #3063 (2nd from bottom), or PI3 Kinase p85 (19H8) Rabbit mAb #4257 (bottom). (Data kindly provided by Drs. Rebecca Chin and Alex Toker, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA).

转染了表达Akt1的shRNA或Akt2 ShRNA的质粒,不诱导(-)或doxycycline诱导指定时间后的LNCaP细胞提取物,使用Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb(Akt1特异)#9018(上),Phospho-Akt2 (Ser474) (D3H2) Rabbit mAb (Akt2特异)(从上的第二道),Akt1 (C73H10) Rabbit mAb#2938(中),Akt2 (D6G4) Rabbit mAb #3063(从下的第二道),或PI3 Kinase p85 (19H8) Rabbit mAb #4257(底部)进行western blot分析。(数据由Beth Israel Deaconess Medical Center和Harvard Medical School, Boston, MA的Drs. Rebecca Chin以及Alex Toker友情提供)。

Western Blotting

Western Blotting

Western blot analysis of purified recombinant phospho-Akt1, phospho-Akt2 and phospho-Akt3 proteins using Phospho-Akt1 (Ser473) (D7F10) XP®Rabbit mAb (Akt1 Specific) (upper), Phospho-Akt (Ser473) (D9E) XP®Rabbit mAb #4060 (middle) and Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

对纯化的重组phospho-Akt1, phospho-Akt2 和phospho-Akt3,使用Phospho-Akt1 (Ser473) (D7F10) XP®Rabbit mAb (Akt1 Specific)(上),Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060(中)Akt (pan) (C67E7) Rabbit mAb#4691(下)进行western blot分析。

Background

Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 (15) and p21 Waf1/Cip1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18,19).

Akt,又被称为PKB 或 Rac,在细胞的生长和凋亡中起到关键作用(1-3)。该蛋白激酶可以被胰岛素和多种生长和存活因子激活,在涉及PI3K激酶的wortmannin(渥曼青霉素)敏感信号通路中发挥作用(2,3)。Akt可由磷脂结合激活,该过程通过活化环中的Thr308 (4)位点以及羧基端Ser473位点的磷酸化完成,其中Thr308的磷酸化由PDK1完成。 之前推测PDK2在Ser473位点磷酸化Akt,后被证实为哺乳动物rapamycin(雷帕霉素)靶蛋白mTOR 的作用, 它存在在一个含有rictor 和Sin1 的rapamycin(雷帕霉素)非敏感复合体中。Akt促进细胞的生长通过抑制细胞的凋亡 ,例如Akt可以抑制下游靶蛋白Bad (7), forkhead 转录因子 (8), c-Raf (9), and caspase-9。PTEN是PI3K激酶/Akt信号通路的主要负调控因子(10)。 LY294002是特异性的 PI3K 激酶抑制剂(11)。Akt 的另外一个主要功能是通过磷酸化并进而抑制GSK-3α 和 β来调控糖原的合成(12,13)。 Akt 也可以调控胰岛素(insulin)诱导的葡萄糖转运 (12)。除此之外,Akt还可以调控细胞周期,这个功能通过抑制GSK-3β,从而调控其下游的cyclin D1 的磷酸化和降解 (14),或者负向调控cyclin依赖的激酶抑制因子 p27 Kip (15) 和 p21 Waf1/CIP1 (16)来实现。 Akt 也可以通过直接磷酸化含有raptor的rapamycin(雷帕霉素)敏感复合体中的mTOR来调控细胞的生长(17)。 更重要的是, Akt磷酸化和抑制TSC2, 而TCS2是mTOR-raptor复合物中mTOR的抑制因子(18,19)。

  1. Franke, T.F. et al. (1997) Cell 88, 435-7.
  2. Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.
  3. Franke, T.F. et al. (1995) Cell 81, 727-36.
  4. Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.
  5. Sarbassov, D.D. et al. (2005) Science 307, 1098-101.
  6. Jacinto, E. et al. (2006) Cell 127, 125-37.
  7. Cardone, M.H. et al. (1998) Science 282, 1318-21.
  8. Brunet, A. et al. (1999) Cell 96, 857-68.
  9. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
  10. Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.
  11. Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.
  12. Hajduch, E. et al. (2001) FEBS Lett 492, 199-203.
  13. Cross, D.A. et al. (1995) Nature 378, 785-9.
  14. Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511.
  15. Gesbert, F. et al. (2000) J Biol Chem 275, 39223-30.
  16. Zhou, B.P. et al. (2001) Nat Cell Biol 3, 245-52.
  17. Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.
  18. Inoki, K. et al. (2002) Nat Cell Biol 4, 648-57.
  19. Manning, B.D. et al. (2002) Mol Cell 10, 151-62.

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