| 货号 | 7272S |
| 反应种属 | Human/Mouse |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Signaling Nodes Multi-Target Sandwich ELISA Kit #7272 detects endogenous levels of six proteins: Akt1, phospho-Akt1 (Ser473), phospho-MEK1 (Ser217/221), phospho-p38 MAPK (Thr180/Tyr182), phospho-Stat3 (Tyr705) and phospho-NF-κB p65 (Ser536). Differential phosphorylation of these proteins can be observed over time in response to various growth factor and cytokine treatments, as shown in Figure 1. |
| 供应商 | CST |
| 背景 | Akt is a protooncogene with a critical regulatory role in diverse cellular processes including growth, survival and the cell cycle. Akt is also a major regulator of insulin signaling and glucose metabolism (1-4). Akt is activated by PI3 kinase signaling and activation loop phosphorylation at Thr308 by PDK1 and by phosphorylation withing the carboxyl terminus at Ser473 by PDK2 (5-7). MEK1 and MEK2 are dual-specificity protein kinases that function in a mitogen activated protein kinase cascade controlling cell growth and differentiation. Activation of MEK1 and MEK2 occurs through phosphorylation of serine 217 and serine 221 by Raf-like molecules. MEK activates p44 and p42 MAP kinase (8-10). p38 MAP kinase (MAPK) participates in a signaling cascade controlling the cellular response to pro-inflammatory cytokines and a variety of cellular stresses. MKK3, MKK6 and SEK (MKK4) activate p38 MAP kinase by phosphorylation at Thr180 and Tyr182 (11-14). The Stat3 transcription factor is an important signaling molecule for many cytokines and growth factor receptors. Stat3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation and DNA binding (15,16). Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammation, stress and immune responses. There are five family members in mammals: RelA/p65, c-Rel, RelB, NF-κB1 (p105/p50) and NF-κB2 (p100/p52). These proteins function as dimeric transcription factors. In unstimulated cells, NF-κB/Rel proteins are sequestered in the cytoplasm and inhibited by the IκB proteins. NF-κB-activating agents induce phosphorylation of IκBs, targeting them for degradation and thereby releasing the NF-κB/Rel complexes. Active NF-κB/Rel complexes are further activated by phosphorylation (17-20). |
| 存放说明 | 4C |
| 参考文献 | 1 . Kim, D. and Chung, J. (2002) J Biochem Mol Biol 35, 106-15. 2 . Manning, B.D. and Cantley, L.C. (2007) Cell 129, 1261-74. 3 . McKay, M.M. and Morrison, D.K. (2007) Oncogene 26, 3113-21. 4 . Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44. 5 . Zdychová, J. and Komers, R. (2005) Physiol Res 54, 1-16. 6 . Ghosh, S. and Karin, M. (2002) Cell 109, S81-S96. 7 . DiDonato, J. et al. (1996) Mol Cell Biol 16, 1295-304. 8 . Song, G. et al. J Cell Mol Med 9, 59-71. 9 . Sakurai, H. et al. (1999) J Biol Chem 274, 30353-6. 10 . Alessi, D.R. et al. (1996) EMBO J 15, 6541-51. 11 . Sarbassov, D.D. et al. (2005) Science 307, 1098-101. 12 . Raingeaud, J. et al. (1995) J. Biol. Chem. 270, 7420-7426. 13 . Jacinto, E. et al. (2006) Cell 127, 125-37. 14 . Alessi, D.R. et al. (1994) EMBO J. 13, 1610-19. 15 . Pearson, G. et al. (2001) Endocr Rev 22, 153-83. 16 . Raman, M. et al. (2007) Oncogene 26, 3100-12. 17 . Zarubin, T. and Han, J. (2005) Cell Res 15, 11-8. 18 . OShea, J.J. et al. (2002) Cell 109 Suppl, S121-31. 19 . Kaptein, A. et al. (1996) J Biol Chem 271, 5961-4. 20 . Mattioli, I. et al. (2004) J Immunol 172, 6336-44. |
Figure 1. Treatment of NIH/3T3 cells with PDGF (A), TNFα/IL-1β (B) or IL-6 (C) induces differential phosphorylation of Akt1 at Ser473, Stat3 at Tyr705, p38α MAPK at Thr180/Tyr182, MEK1 at Ser217/221 and NF-κB p65 at Ser536 as detected by the PathScan® Signaling Nodes Multi-Target Sandwich ELISA Kit #7272. While dynamic changes in phosphorylation are observed throughout the time course, the level of total Akt, MEK1, Stat3, p38α MAPK and NF-κB p65 remains unchanged as demonstrated by sandwich ELISA and Western analysis. NIH/3T3 cells (80-90% confluent) were starved overnight and stimulated with either PDGF (100 ng/mL) or IL-6 (100 ng/mL) for 5, 10, 20, 40 and 80 minutes at 37ºC. For simultaneous treatment with TNF-α and IL-1β, exponentially growing cultures of NIH/3T3 (80-90% confluent) were treated for the indicated times at 37ºC with 20 ng/mL TNF-α and 10 ng/mL IL-1β. Lysates were assayed at a protein concentration of 0.45 mg/mL. The absorbance readings at 450 nm are shown as a 3-dimensional representation in the left figure, while the corresponding Western blots are shown in the right figure. The antibodies used for the Western analyses include Akt Antibody #9272, Phospho-Akt (Ser473) (193H12) Rabbit mAb #4058, MEK1 (61B12) Mouse mAb #2352, Phospho-MEK1/2 (Ser217/221) Antibody #9121, Stat3 Antibody #9132, Phospho-Stat3 (Tyr705) (3E2) Mouse mAb #9138, p38 MAP Kinase Antibody #9218, Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mAb #9216, NF-κB p65 Antibody #3034 and Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033. | |
Figure 2. Schematic representation of a 96-well plate depicting the color-code of the reagents used to detect endogenous levels of Akt1 (red; 1 & 2), Phospho-Akt1 (Ser473) (tan; 3 & 4), Phospho-MEK1 (Ser217/221) (green; 5 & 6), Phospho-p38 MAPK (Thr180/Tyr182) (yellow; 7 & 8), Phospho-Stat3 (Tyr705) (dark pink; 9 & 10) and Phospho-NF-κB p65 (Ser536) (orange; 11 & 12).图2：呈现的是96孔板的图解，描述了试剂的颜色代码，用于检测内源性 Akt1 (红色; 1 & 2), Phospho-Akt1 (丝氨酸473位点) (棕褐色; 3 & 4), Phospho-MEK1 (丝氨酸217/221位点) (绿色; 5 & 6), Phospho-p38 MAPK (苏氨酸180位点/酪氨酸182位点) (黄色; 7 & 8), Phospho-Stat3 (Tyr705) (深粉色; 9 & 10)和Phospho-NF-κB p65 (丝氨酸536位点) (橘色; 11 & 12)。 |
