Growth and Differentiation Phosphorylation Sampler Kit

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1 Growth and Differentiation Phosphorylation Sampler Kit E Kits Includes Cat. Quantity Application Reactivity Source Akt (Phospho-Ser473) E μg/50μl IHC, WB Human, Mouse, Rat Rabbit MEK1 (Phospho-Ser221) E μg/50μl IHC, WB Human, Mouse, Rat Rabbit MEK-2 (Phospho-Thr394) E μg/50μl IHC, WB, IF Human, Mouse, Rat Rabbit PDK1 (Phospho-Ser241) E μg/50μl IHC, WB, IF Human, Mouse, Rat Rabbit Raf1 (Phospho-Ser259) E μg/50μl IHC, WB Human, Mouse, Rat Rabbit The serine-threonine protein kinase encoded by the AKT1 gene is catalytically inactive in serum-starved primary and immortalized fibroblasts. AKT1 and the related AKT2 are activated by platelet-derived growth factor. The activation is rapid and specific, and it is abrogated by mutations in the pleckstrin homology domain of AKT1. It was shown that the activation occurs through phosphatidylinositol 3-kinase. In the developing nervous system AKT is a critical mediator of growth factor-induced neuronal survival. Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase AKT1, which then phosphorylates and inactivates components of the apoptotic machinery. Multiple alternatively spliced transcript variants have been found for this gene. General protein kinase capable of phosphorylating several known proteins. Phosphorylates TBC1D4. Signals downstream of phosphatidylinositol 3-kinase (PI(3)K) to mediate the effects of various growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I). Plays a role in glucose transport by mediating insulin-induced translocation of the GLUT4 glucose transporter to the cell surface. Mediates the antiapoptotic effects of IGF-I. Mediates insulin-stimulated protein synthesis by phosphorylating TSC2 at 'Ser-939' and 'Thr-1462', thereby activating mtorc1 signaling and leading to both phosphorylation of 4E-BP1 and in activation of RPS6KB1. Promotes glycogen synthesis by mediating the insulin-induced activation of glycogen synthase. Akt (Protein kinase B, PKB) is a serine/threonine kinase that plays a key in regulating cell survival, insulin signaling, angiogenesis and tumor formation. Akt is a downstream mediator of the PI 3-K pathway, resulting in the recruitment of Akt to the plasma membrane via the PH (plexstrin homology domain) of Akt. Akt is fully activated by phosphorylation at two key sites: Ser308 (phosphorylated by PDK1) and Thr478 (phosphorylated by mtor and DNA-PK). Akt can then phosphorylated a wide range of substrates including transcription factors (e.g. FOXO1), kinases (GSK-3, Raf-1, ASK, Chk1) and other proteins with important signaling roles (e.g. Bad, MDM2). There are three isoforms of Akt; Akt 1, 2 and 3 (also known as PKBalpha, beta and gamma). Phosphorylation of AKT on Thr-308, Ser-473 and Tyr-474 is required for full activity. Ser-473 phosphorylation by mtorc2 favors, Thr-308 phosphorylation by PDPK1. Ser-473 phosphorylation is enhanced by interaction with 1

2 AGAP2 isoform 2 (PIKE-A). Ser-473 phosphorylation is enhanced in focal cortical dysplasias with Taylor-type balloon cells. MAPKSP1 gene encodes a scaffold protein that functions in the extracellular signal-regulated kinase (ERK) cascade. The protein is localized to late endosomes by the mitogen-activated protein-binding protein-interacting protein, and binds specifically to MAP kinase kinase MAP2K1/MEK1, MAP kinase MAPK3/ERK1, and MAP kinase MAPK1/ERK2. Studies of the orthologous gene in mouse indicate that it regulates late endosomal traffic and cell proliferation. Multiple transcript variants are expressed by this gene, but only one variant is thought to express a protein. Adapter protein that enhances the efficiency of the MAP kinase cascade. Facilitates the activation of MAPK2 Pyruvate dehydrogenase (PDH) is a mitochondrial multienzyme complex that catalyzes the oxidative decarboxylation of pyruvate and is one of the major enzymes responsible for the regulation of homeostasis of carbohydrate fuels in mammals. The enzymatic activity is regulated by a phosphorylation/dephosphorylation cycle. Phosphorylation of PDH by a specific pyruvate dehydrogenase kinase (PDK) results in inactivation. Inhibits the mitochondrial pyruvate dehydrogenase complex by phosphorylation of the E1 alpha subunit, thus contributing to the regulation of glucose metabolism RAF1 protein is a MAP kinase kinase kinase (MAP3K), which functions downstream of the Ras family of membrane associated GTPases to which it binds directly. Once activated, the cellular RAF1 protein can phosphorylate to activate the dual specificity protein kinases MEK1 and MEK2, which in turn phosphorylate to activate the serine/threonine specific protein kinases, ERK1 and ERK2. Activated ERKs are pleiotropic effectors of cell physiology and play an important role in the control of gene expression involved in the cell division cycle, apoptosis, cell differentiation and cell migration. Mutations in this gene are associated with Noonan syndrome 5 and LEOPARD syndrome 2. Involved in the transduction of mitogenic signals from the cell membrane to the nucleus. Part of the Ras-dependent signaling pathway from receptors to the nucleus. Protects cells from apoptosis mediated by STK3. Raf kinases, a family of three serine/threonine kinases, are part of the ras-mapk signaling cascade and phosphorylate MEK. Upon growth factor stimulation, Raf-1 (or c-raf) is activated by GTP-bound Ras and recruited to the cell membrane. This activation process is tightly regulated by a number of factors including phosphatases (e.g. PP1, PP2A, PP5), kinases (e.g. Src, ERK, Akt, PKC) and proteins that bind directly to Raf-1 (e.g. RKIP, zeta, KSR, Hsp90). Raf-1 is also thought to be able to dimerize with wild type B-Raf in a Ras-dependent process. B-raf is commonly mutated and thereby activated in many human cancers, the most frequent mutation being the V600E mutation of the kinase domain. Whilst wt b-raf and Raf-1 are strongly activated by growth factor signals via Ras and Src, a-raf is only modestly activated and has low basal activity. All three isoforms of Raf are considered to be oncogenic. 2

3 Catalog Number: E , E Amount: Form of : Storage/Stability: Immunogen: Purification: Specificity/Sensitivity: Reactivity: 50μg/50μl, 100μg/100μl Rabbit IgG in phosphate buffered saline (without Mg 2+ and Ca 2+ ), ph 7.4, 150mM NaCl, Store at -20 /1 year The antiserum was produced against synthesized phosphopeptide derived from human Akt around the phosphorylation site of serine 473 (Q-F-S P -Y-S). The antibody was affinity-purified from rabbit antiserum by affinity-chromatography using Akt (phospho-ser473) antibody detects endogenous levels of Akt only when phosphorylated at serine 473. Human, Mouse, Rat Applications: WB: 1:500~1:1000 IHC: 1:50~1:100 Swiss-Prot No. : P31749 Akt (Phospho-Ser473) References: Baudhuin LM, et al. (2004) FASEB J Feb; 18(2): Min YH, et al. (2004) Cancer Res; 64(15): Feng J, et al. (2004) J Biol Chem; 279(34): E Immunohistochemical analysis of paraffin- embedded human breast carcinoma tissue, using Akt (phospho-ser473) antibody (E011054). Western blot analysis of extract from HeLa cells untreated or treated with heat shock using Akt (Ab-473) antibody (E021054, Lane 1 and 2) and Akt (phospho-ser473) antibody (E011054, Lane 3 and 4). 3

4 MEK1 (Phospho-Ser221) E Catalog Number: E , E Form of : Rabbit IgG in phosphate buffered saline (without Mg 2+ and Ca 2+ ), ph 7.4, 150mM NaCl, MEK1 around the phosphorylation site of serine 221 (A-N-S P -F-V). Specificity/Sensitivity: MEK1 (phospho-ser221) antibody detects endogenous levels of MEK1 only when phosphorylated at serine 221. Applications: WB: 1:500~1:1000 IHC: 1:50~1:100 Swiss-Prot No. : Q02750 References: Zebisch A, et al. (2006) Cancer Res; 66(7): Luciano BS, et al.(2006)j Biol Chem; 279(50): Wang X, et al. (2003) Oncogene; 22(1): Gopalbhai K, et al. (2003) J Biol Chem; 278(10): Ling MT, et al. (2002)Oncogene; 21(55): Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using MEK1 (phospho- Ser221) antibody (E011161). Western blot analysis of extracts from Jurkat cells, using MEK1 (Ab-221) antibody (E021175, Lane 1 and 2) and MEK1 (phospho-ser221) antibody (E011161, Lane 3 and 4). UV Peptide

5 MEK2 (Phospho-Thr394) E Catalog Number: E , E Form of : Rabbit IgG in phosphate buffered saline (without Mg 2+ and Ca 2+ ), ph 7.4, 150mM NaCl, MEK2 around the phosphorylation site of threonine 394 (P-G-T P -P-T). Specificity/Sensitivity: MEK2 (phospho-thr394) antibody detects endogenous levels of MEK2 only when phosphorylated at threonine 394. Applications: WB: 1:500~1:1000 IHC: 1:50~1:100 IF: 1: Swiss-Prot No. : P36507 References: Crews C M, et al. (1992) Science. 258: Alessi D R, et al. (1994) EMBO J. 13: Rosen L B, et al. (1994) Neuron. 12: Immunohistochemical analysis of paraffin- embedded human breast carcinoma tissue using MEK2 (phospho-thr394) antibody (E011008). Peptide P-Peptide Western blot analysis of extracts from ovary cancer cells using MEK2 (phospho-thr394) antibody (E011008). 5

6 PDK1 (Phospho-Ser241) E Catalog Number: E , E Form of : Rabbit IgG in phosphate buffered saline (without Mg 2+ and Ca 2+ ), ph 7.4, 150mM NaCl, PDK1 around the phosphorylation site of serine 241 (A-N-S P -F-V). Specificity/Sensitivity: PDK1 (phospho-ser241) antibody detects endogenous levels of PDK1 only when phosphorylated at serine 241. Applications: WB: 1:500~1:1000 IHC: 1:50~1:100 IF: 1: Swiss-Prot No. : O15530 References: Scheid MP,et al. (2005)Mol Cell Biol; 25(6): Chen H, et al. (2001) Biochemistry; 40(39): Sato S,et al. (2002) J Biol Chem; 277(42): Lim MA, et al.(2003)proc Natl Acad Sci U S A; 100(24): Immunohistochemical analysis of paraffin- embedded human breast carcinoma tissue using PDK1 (phospho-ser241) antibody (E011005). EGF - + Western blot analysis of extracts from MDA-MB-435 cells using PDK1 (phospho-ser241) antibody (E011005). 6

7 Raf-1 (Phospho-Ser259) E Catalog Number: E , E Form of : Rabbit IgG in phosphate buffered saline (without Mg 2+ and Ca 2+ ), ph 7.4, 150mM NaCl, Raf-1 around the phosphorylation site of serine 259 (S-T-S P -T-P). Specificity/Sensitivity: Raf-1 (phospho-ser259) antibody detects endogenous levels of Raf-1 only when phosphorylated at serine 259. Applications: WB: 1:500~1:1000 IHC: 1:50~1:100 Swiss-Prot No. : P04049 References: Dougherty M K, et al. (2005) Mol Cell. 17(2): Hekman M, et al. (2005) FEBS Lett. 579(2): Avruch J,et al. (1994) Trends Biochem. Sci. 19, Chong H, et al. (2001) EMBO J. 20, King A J, et al. (1998) Nature. 396: Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using Raf-1 (phospho-ser259) antibody (E011006). K562 MDA-MB-435 K562 Peptide P-Peptide Western blot analysis using Raf-1 (Ab-259) antibody (E021006, Line 1, 2, and 3) and Raf-1 (phospho- Ser259) antibody (E011006, Line 4, 5, and 6). 7

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