Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M||WB||Rb||Purified||Monoclonal Antibody|
|Description||Anti-phospho-Akt1/PKBα (Ser473) Antibody, clone SK703, rabbit monoclonal|
|Presentation||Protein A purified antibody in 0.1M Tris-glycine, pH 7.4, 0.15M NaCl, 0.05% sodium azide.|
|Application||Detect phospho-Akt1/PKBα (Ser473) using this Anti-Akt1 Antibody, clone SK703 validated for use in WB.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable refrigerated at 2-8°C in undiluted aliquots for up to 1 year from date of receipt.|
|Material Size||100 µg|
|Reference overview||Pub Med ID|
|RLIP76 regulates PI3K/Akt signaling and chemo-radiotherapy resistance in pancreatic cancer.|
Leake, K; Singhal, J; Nagaprashantha, LD; Awasthi, S; Singhal, SS
PloS one 7 e34582 2012
Pancreatic cancer is an aggressive malignancy with characteristic metastatic course of disease and resistance to conventional chemo-radiotherapy. RLIP76 is a multi-functional cell membrane protein that functions as a major mercapturic acid pathway transporter as well as key regulator of receptor-ligand complexes. In this regard, we investigated the significance of targeting RLIP76 on PI3K/Akt pathway and mechanisms regulating response to chemo-radiotherapy.Cell survival was assessed by MTT and colony forming assays. Cellular levels of proteins and phosphorylation was determined by Western blot analyses. The impact on apoptosis was determined by TUNEL assay. The anti-cancer effects of RLIP76 targeted interventions in vivo were determined using mice xenograft model of the pancreatic cancer. The regulation of doxorubicin transport and radiation sensitivity were determined by transport studies and colony forming assays, respectively.Our current studies reveal an encompassing model for the role of RLIP76 in regulating the levels of fundamental proteins like PI3K, Akt, E-cadherin, CDK4, Bcl2 and PCNA which are of specific importance in the signal transduction from critical upstream signaling cascades that determine the proliferation, apoptosis and differentiation of pancreatic cancer cells. RLIP76 depletion also caused marked and sustained regression of established human BxPC-3 pancreatic cancer tumors in nude mouse xenograft model. RLIP76 turned out to be a major regulator of drug transport along with contributing to the radiation resistance in pancreatic cancer.RLIP76 represents a mechanistically significant target for developing effective interventions in aggressive and refractory pancreatic cancers.
|Resveratrol helps recovery from fatty liver and protects against hepatocellular carcinoma induced by hepatitis B virus X protein in a mouse model.|
Lin, HC; Chen, YF; Hsu, WH; Yang, CW; Kao, CH; Tsai, TF
Cancer prevention research (Philadelphia, Pa.) 5 952-62 2012
Resveratrol is a natural polyphenol that has beneficial effects across species and various disease models. Here, we investigate whether resveratrol is effective against hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) using HBV X protein (HBx) transgenic mice. We found that resveratrol (30 mg/kg/d) has a therapeutic effect on HBx-induced fatty liver and the early stages of liver damage. Resveratrol decreased intracellular reactive oxygen species and transiently stimulated hepatocyte proliferation. Interestingly, resveratrol inhibited LXRα and downregulated the expression of the lipogenic genes, Srebp1-c and PPARγ. The decrease in Srebp1-c seems to further downregulate the expression of its target genes, Acc and Fas. In addition, resveratrol stimulated the activity of Ampk and SirT1. Thus, resveratrol has a pleiotropic effect on HBx transgenic mice in terms of the downregulation of lipogenesis, the promotion of transient liver regeneration, and the stimulation of antioxidant activity. Furthermore, at the later precancerous stages, resveratrol delayed HBx-mediated hepatocarcinogenesis and reduced HCC incidence from 80% to 15%, a 5.3-fold reduction. Resveratrol should be considered as a potential chemopreventive agent for HBV-associated HCC.
|Calyculin A from Discodermia calyx is a dual action toxin that blocks calcium influx and inhibits protein Ser/Thr phosphatases.|
Holy, M; Brautigan, DL
Toxins 4 940-54 2012
Calyculin A (Caly A) is cell permeable toxin widely used in cell biology research as an inhibitor of type 1 and type 2A protein Ser/Thr phosphatases of the PPP family. Here we tested effects of low concentrations of Caly A on proliferation of human cancer and non-cancer cell lines. We found that long-term 0.3 nM Caly A prevented G1 to S phase cell cycle progression in human Hs-68 fibroblasts and ARPE19 epithelial cells, but not human breast cancer MDA-MB-468, MDA-MB-231 and MCF7 cells. These conditions produced no change in cyclin D1 levels or in the phosphorylation of endogenous proteins. However, acute application of 0.3 nM Caly A blocked serum-induced increase in intracellular calcium levels in Hs-68 fibroblasts, but not in MDA-MB-468 breast cancer cells. We propose that subnanomolar Caly A prevents cell cycle progression because it blocks calcium uptake by fibroblasts. This probably involves non-selective cation channels and cancer cell proliferation was not affected because calcium enters these cells by other channels. Our results suggest that calyculin A has dual actions and acts as a channel blocker, in addition to its well-established effects as a phosphatase inhibitor.
|The ATM kinase signaling induced by the low-energy β-particles emitted by (33)P is essential for the suppression of chromosome aberrations and is greater than that induced by the energetic β-particles emitted by (32)P.|
White JS, Yue N, Hu J, Bakkenist CJ
Mutation research 708 28-36. Epub 2011 Feb 16. 2011
Ataxia-telangiectasia mutated (ATM) encodes a nuclear serine/threonine protein kinase whose activity is increased in cells exposed to low doses of ionizing radiation (IR). Here we examine ATM kinase activation in cells exposed to either (32)P- or (33)P-orthophosphate under conditions typically employed in metabolic labelling experiments. We calculate that the absorbed dose of IR delivered to a 5cm×5cm monolayer of cells incubated in 2ml media containing 1mCi of the high-energy (1.70MeV) β-particle emitter (32)P-orthophosphate for 30min is ∼1Gy IR. The absorbed dose of IR following an otherwise identical exposure to the low-energy (0.24MeV) β-particle emitter (33)P-orthophosphate is ∼0.18Gy IR. We show that low-energy β-particles emitted by (33)P induce a greater number of ionizing radiation-induced foci (IRIF) and greater ATM kinase signaling than energetic β-particles emitted by (32)P. Hence, we demonstrate that it is inappropriate to use (33)P-orthophosphate as a negative control for (32)P-orthophosphate in experiments investigating DNA damage responses to DNA double-strand breaks (DSBs). Significantly, we show that ATM accumulates in the chromatin fraction when ATM kinase activity is inhibited during exposure to either radionuclide. Finally, we also show that chromosome aberrations accumulate in cells when ATM kinase activity is inhibited during exposure to ∼0.36Gy β-particles emitted by (33)P. We therefore propose that direct cellular exposure to (33)P-orthophosphate is an excellent means to induce and label the IR-induced, ATM kinase-dependent phosphoproteome.Copyright © 2011 Elsevier B.V. All rights reserved.
|Cereal and nonfat milk support muscle recovery following exercise.|
Lynne Kammer, Zhenping Ding, Bei Wang, Daiske Hara, Yi-Hung Liao, John L Ivy, Lynne Kammer, Zhenping Ding, Bei Wang, Daiske Hara, Yi-Hung Liao, John L Ivy
Journal of the International Society of Sports Nutrition 6 11 2009
ABSTRACT: BACKGROUND: This study compared the effects of ingesting cereal and nonfat milk (Cereal) and a carbohydrate-electrolyte sports drink (Drink) immediately following endurance exercise on muscle glycogen synthesis and the phosphorylation state of proteins controlling protein synthesis: Akt, mTOR, rpS6 and eIF4E. METHODS: Trained cyclists or triathletes (8 male: 28.0 +/- 1.6 yrs, 1.8 +/- 0.0 m, 75.4 +/- 3.2 kg, 61.0 +/- 1.6 ml O2*kg-1*min-1; 4 female: 25.3 +/- 1.7 yrs, 1.7 +/- 0.0 m, 66.9 +/- 4.6 kg, 46.4 +/- 1.2 mlO2*kg-1*min-1) completed two randomly-ordered trials serving as their own controls. After 2 hours of cycling at 60-65% VO2MAX, a biopsy from the vastus lateralis was obtained (Post0), then subjects consumed either Drink (78.5 g carbohydrate) or Cereal (77 g carbohydrate, 19.5 g protein and 2.7 g fat). Blood was drawn before and at the end of exercise, and at 15, 30 and 60 minutes after treatment. A second biopsy was taken 60 minutes after supplementation (Post60). Differences within and between treatments were tested using repeated measures ANOVA. RESULTS: At Post60, blood glucose was similar between treatments (Drink 6.1 +/- 0.3, Cereal 5.6 +/- 0.2 mmol/L, p .05), but after Cereal, plasma insulin was significantly higher (Drink 123.1 +/- 11.8, Cereal 191.0 +/- 12.3 pmol/L, p .05), and plasma lactate significantly lower (Drink 1.4 +/- 0.1, Cereal 1.00 +/- 0.1 mmol/L, p .05). Except for higher phosphorylation of mTOR after Cereal, glycogen and muscle proteins were not statistically different between treatments. Significant Post0 to Post60 changes occurred in glycogen (Drink 52.4 +/- 7.0 to 58.6 +/- 6.9, Cereal 58.7 +/- 9.6 to 66.0 +/- 10.0 mumol/g, p .05) and rpS6 (Drink 17.9 +/- 2.5 to 35.2 +/- 4.9, Cereal 18.6 +/- 2.2 to 35.4 +/- 4.4 %Std, p .05) for each treatment, but only Cereal significantly affected glycogen synthase (Drink 66.6 +/- 6.9 to 64.9 +/- 6.9, Cereal 61.1 +/- 8.0 to 54.2 +/- 7.2%Std, p .05), Akt (Drink 57.9 +/- 3.2 to 55.7 +/- 3.1, Cereal 53.2 +/- 4.1 to 60.5 +/- 3.7 %Std, p .05) and mTOR (Drink 28.7 +/- 4.4 to 35.4 +/- 4.5, Cereal 23.0 +/- 3.1 to 42.2 +/- 2.5 %Std, p .05). eIF4E was unchanged after both treatments. CONCLUSION: These results suggest that Cereal is as good as a commercially-available sports drink in initiating post-exercise muscle recovery.Full Text Article
|Inducible-NOS but not neuronal-NOS participate in the acute effect of TNF-alpha on hypothalamic insulin-dependent inhibition of food intake.|
Juliana C Moraes, Maria E Amaral, Paty K Picardi, Vivian C Calegari, Talita Romanatto, Marcela Bermúdez-Echeverry, Silvana Chiavegatto, Mario J Saad, Licio A Velloso
FEBS letters 580 4625-31 2006
TNF-alpha acts on the hypothalamus modulating food intake and energy expenditure through mechanisms incompletely elucidated. Here, we explore the hypothesis that, to modulate insulin-induced anorexigenic signaling in hypothalamus, TNF-alpha requires the synthesis of NO. TNF-alpha activates signal transduction through JNK and p38 in hypothalamus, peaking at 10(-8) M. This is accompanied by the induction of expression of the inducible and neuronal forms of NOS, in both cases peaking at 10(-12) M. In addition, TNF-alpha stimulates NOS catalytic activity. Pre-treatment with TNF-alpha at a low dose (10(-12) M) inhibits insulin-dependent anorexigenic signaling, and this effect is abolished in iNOS but not in nNOS knockout mice.
|3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro.|
Alessi, D R, et al.
Curr. Biol., 8: 69-81 (1998) 1998
|PDK1, one of the missing links in insulin signal transduction?|
Cohen, P, et al.
FEBS Lett., 410: 3-10 (1997) 1997
The initial steps in insulin signal transduction occur at the plasma membrane and lead to the activation of phosphatidylinositide (PtdIns) 3-kinase and the formation of PtdIns(3,4,5,)P3 in the inner leaflet of the plasma membrane which is then converted to PtdIns(3,4)P2 by a specific phosphatase. Inhibitors of PtdIns 3-kinase suppress nearly all the metabolic actions of insulin indicating that PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2 are key 'second messengers' for this hormone. A major effect of insulin is its ability to stimulate the synthesis of glycogen in skeletal muscle. By 'working backwards' from glycogen synthesis, we have dissected an insulin-stimulated protein kinase cascade which is triggered by the activation of PtdIns 3-kinase. The first enzyme in this cascade is termed 3-phosphoinositide-dependent protein kinase (PDK1), because it is only active in the presence of PtdIns(3,4,5)P3 or PtdIns(3,4)P2. PDK1 then activates protein kinase B (PKB) which, in turn, inactivates glycogen synthase kinase-3 (GSK3), leading to the dephosphorylation and activation of glycogen synthase and hence to an acceleration of glycogen synthesis. We review the evidence which indicates that the phosphorylation of other proteins by PKB and GSK3 is likely to mediate many of the intracellular actions of insulin.
|3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase.|
Alessi, D R, et al.
Curr. Biol., 7: 776-89 (1997) 1997
|Specific binding of the Akt-1 protein kinase to phosphatidylinositol 3,4,5-trisphosphate without subsequent activation.|
James, SR; Downes, CP; Gigg, R; Grove, SJ; Holmes, AB; Alessi, DR
The Biochemical journal 315 ( Pt 3) 709-13 1996
Recent evidence has suggested that activation of phosphoinositide 3-kinase (PI 3-kinase) is required for the activation of Akt-1 by growth factors and insulin. Here we demonstrate by two independent methods that Akt-1 from L6 myotubes binds to PtdIns(3,4,5)P3, PtdIns(3,4)P2 and PtdIns(4,5)P2 when presented against a background of phosphatidylserine (PtdSer) or a 1:1 mixture of PtdSer and phosphatidylcholine (PtdCho). No binding was observed with the lipids PtdIns(3,5)P2, PtdIns4P and PtdIns3P or background lipids. Activated, hyperphosphorylated forms of Akt-1 from insulin-stimulated L6 myotubes bound to PtdIns(3,4,5)P3 in a similar manner as inactive Akt-1. Quantitative analysis using surface plasmon resonance showed that the equilibrium association constant for the binding of Akt-1 to PtdIns(3,4,5)P3 was submicromolar and that PtdIns(3,4)P2 and PtdIns(4,5)P2 bound to Akt-1 with 3- and 6-fold lower affinities respectively. Interaction of Akt-1 with PtdIns(3,4,5)P3 did not activate the protein kinase activity, either before or after incubation with MgATP. A model is presented in which PtdIns(3,4,5)P3 may prime Akt-1 for activation by another protein kinase, perhaps by recruiting it to the plasma membrane.