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產(chǎn)品編號	bs-0876R
英文名稱	Rabbit　Anti-phospho-AKT (Ser473)　antibody
中文名稱	磷酸化蛋白激酶B抗體
別　　　　名	Akt(Phospho-Ser473); AKT (phospho-S473); AKT (phospho Ser473); p-AKT (Ser473); AKT 1; AKT; AKT1; AKT-1; AKT1_HUMAN; C AKT; cAKT; MGC9965; MGC99656; Oncogene AKT1; PKB; PKB alpha; PKB-ALPHA; PRKBA; Protein Kinase B Alpha; Protein kinase B; Proto-oncogene c-Akt; RAC Alpha; RAC alpha serine/threonine protein kinase; RAC; RAC PK Alpha; Rac protein kinase alpha; RAC Serine/Threonine Protein Kinase; RAC-alpha serine/threonine-protein kinase; RAC-PK-alpha; v akt murine thymoma viral oncogene homolog 1; vAKT Murine Thymoma Viral Oncogene Homolog 1.
	Specific References　　(68)　　　　　\|　　　　　bs-0876R has been referenced in 68 publications. [IF=8.469]　Que, Tianshi. et al. HMGA1 stimulates MYH9-dependent ubiquitination of GSK-3β via PI3K/Akt/c-Jun signaling to promote malignant progression and chemoresistance in gliomas. Cell Death Dis. 2021 Dec;12(12):1-12　　WB　;　　Human.　　 PubMed:34887392 [IF=7.561]　Sun J. et al. Plasma Exosomes Transfer miR-885-3p Targeting the AKT/NFκB Signaling Pathway to Improve the Sensitivity of Intravenous Glucocorticoid Therapy Against Graves Ophthalmopathy.. Front Immunol. 2022 Feb;13:819680-819680　　WB　;　　Mouse.　　 PubMed:35265076 [IF=7.129]　Yanwen Hou. et al. Prenatal PM2.5 exposure contributes to neuronal tau lesion in male offspring mice through mitochondrial dysfunction-mediated insulin resistance. ECOTOX ENVIRON SAFE. 2022 Nov;246:114151　　WB　;　　Mouse.　　 PubMed:36228359 [IF=6.67]　Santos, Joana, et al. "Uncovering potential downstream targets of oncogenic GRPR overexpression in prostate carcinomas harboring ETS rearrangements." Oncoscience 2.5 (2015): 497.　　WB　;　　Human.　　 PubMed:26097883 [IF=6.551]　Wei J et al. Endosulfan induces cardiotoxicity through apoptosis via unbalance of pro-survival and mitochondrial-mediated apoptotic pathways. Sci Total Environ. 2020 Jul 20;727:138790.　　WB　;　　human.　　 PubMed:32344260 [IF=6.543]　Wang Jun-wu. et al. 1,25(OH)2D3 Mitigates Oxidative Stress-Induced Damage to Nucleus Pulposus-Derived Mesenchymal Stem Cells through PI3K/Akt Pathway. Oxid Med Cell Longev. 2022;2022:1427110　　IF　;　　Rat.　　 PubMed:35340208 [IF=6.304]　Yunfan Luo. et al. Foxq1 promotes metastasis of nasopharyngeal carcinoma by inducing vasculogenic mimicry via the EGFR signaling pathway. Cell Death Dis. 2021 Apr;12(5):1-16　　WB　;　　Human.　　 PubMed:33875643 [IF=6.023]　Ling Xie. et al. Suppression of GOLM1 by EGCG through HGF/HGFR/AKT/GSK-3β/β-catenin/c-Myc signaling pathway inhibits cell migration of MDA-MB-231. Food Chem Toxicol. 2021 Nov;157:112574　　WB　;　　human.　　 PubMed:34536514 [IF=5.988]　Dongfang Lv. et al. Mailuoshutong pill for varicocele-associated male infertility-Phytochemical characterisation and multitarget mechanism.. FRONT PHARMACOL. 2022 Sep;13:961011-961011　　WB　;　　Rat.　　 PubMed:36160417 [IF=5.895]　Bendong Yang. et al. Naringenin Ameliorates Hyperuricemia by Regulating Renal Uric Acid Excretion via the PI3K/AKT Signaling Pathway and Renal Inflammation through the NF-κB Signaling Pathway. J AGR FOOD CHEM. 2022;XXXX(XXX):XXX-XXX　　WB　;　　Mouse, Human.　　 PubMed:36525382 [IF=5.89]　Shilin Zhang. et al. CD73-Positive Small Extracellular Vesicles Derived From Umbilical Cord Mesenchymal Stem Cells Promote the Proliferation and Migration of Pediatric Urethral Smooth Muscle Cells Through Adenosine Pathway. FRONT BIOENG BIOTECH. 2022; 10: 895998　　WB　;　　Human.　　 PubMed:35573239 [IF=5.81]　Yuqiao Yang. et al. Oxytocin Protects Against Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting PI3K/AKT Pathway via a lncRNA GAS5/miR-375-3p/KLF4-Dependent Mechanism. Front Pharmacol. 2021; 12: 766024　　WB　;　　Rat.　　 PubMed:34925023 [IF=5.715]　　　WB　;　　fish.　　 PubMed:32818499 [IF=5.714]　Zhao H et al. The cardiotoxicity of the common carp (Cyprinus carpio) exposed to environmentally relevant concentrations of arsenic and subsequently relieved by zinc supplementation. Environmental Pollution.2019 Oct; 253:741-748.　　WB　;　　Carp.　　 PubMed:doi:10.1016/j.envpol.2019.07.065 [IF=5.455]　Liu, Qingwu. et al. Huiyang Shengji decoction promotes wound healing in diabetic mice by activating the EGFR/PI3K/ATK pathway. Chin Med-Uk. 2021 Dec;16(1):1-17　　WB　;　　Mouse.　　 PubMed:34727961 [IF=5.246]　Yin Zhuang. et al. Exosomes Secreted by Nucleus Pulposus Stem Cells Derived From Degenerative Intervertebral Disc Exacerbate Annulus Fibrosus Cell Degradation via Let-7b-5p. Front Mol Biosci. 2021; 8: 766115　　WB　;　　Human.　　 PubMed:35111808 [IF=5.195]　Fang Cao. et al. Ginkgo biloba L. extract prevents steroid-induced necrosis of the femoral head by rescuing apoptosis and dysfunction in vascular endothelial cells via the PI3K/AKT/eNOS pathway. J ETHNOPHARMACOL. 2022 Jun;:115476　　WB　;　　Mouse.　　 PubMed:35724747 [IF=5.17]　Liang et al. Histone demethylase RBP2 induced by Helicobactor Pylori CagA participates in the malignant transformation of gastric epithelial cells. (2014) Oncotarge. 5:5798-807　　WB　;　　Human.　　 PubMed:25015565 [IF=5.168]　Ying Gao. et al. ZYY-B-2, a novel ALK inhibitor, overcomes resistance to ceritinib by inhibiting P-gp function and induces apoptosis through mitochondrial pathway in ceritinib-resistant H2228?cells. CHEM-BIOL INTERACT. 2023 Jul;379:110516　　WB　;　　Human.　　 PubMed:37116853 [IF=4.994]　Xinping Song. et al. Lactobacillus plantarum DP189 prevents cognitive dysfunction in D-galactose/AlCl3 induced mouse model of Alzheimer’s disease via modulating gut microbiota and PI3K/Akt/GSK-3β signaling pathway. 2021 Nov 10　　WB　;　　Mouse.　　 PubMed:34755592 [IF=4.932]　Qingyu Meng. et al. Morin hydrate inhibits atherosclerosis and LPS-induced endothelial cells inflammatory responses by modulating the NFκB signaling-mediated autophagy. Int Immunopharmacol. 2021 Nov;100:108096　　WB　;　　Human.　　 PubMed:34464886 [IF=4.932]　Leiying Zhang. et al. Exosomal microRNA-98-5p from hypoxic bone marrow mesenchymal stem cells inhibits myocardial ischemia–reperfusion injury by reducing TLR4 and activating the PI3K/Akt signaling pathway. Int Immunopharmacol. 2021 Dec;101:107592　　WB　;　　Rat.　　 PubMed:34715573 [IF=4.848]　Beibei Chen. et al. Knockdown of Kremen2 Inhibits Tumor Growth and Migration in Gastric Cancer. Front Oncol. 2020; 10: 534095　　WB　;　　Human.　　 PubMed:33489867 [IF=4.831]　Chen Y et al. Epothilone B prevents lipopolysaccharide-induced inflammatory osteolysis through suppressing osteoclastogenesis via STAT3 signaling pathway. Aging (Albany NY). 2020 Jun 11;12(12):11698-11716.　　WB　;　　Mouse.　　 PubMed:32527985 [IF=4.556]　Myoung Su Choi. et al. Activation of the Complement System on Human Endothelial Cells by Urban Particulate Matter Triggers Inflammation-Related Protein Production. Int J Mol Sci. 2021 Jan;22(7):3336　　WB　;　　Human.　　 PubMed:33805189 [IF=4.546]　Huang, Wenjie. et al. The inhibitory effect and mechanism of Yi-qi-hua-yu-jie-du decoction on the drug resistance of gastric cancer stem cells based on ABC transporters. CHIN MED-UK. 2022 Dec;17(1):1-18　　WB　;　　Human.　　 PubMed:35941687 [IF=4.319]　Sihua Wen. et al. Investigating the Mechanism of Action of Schisandra chinensis Combined with Coenzyme Q10 in the Treatment of Heart Failure Based on PI3K-AKT Pathway. DRUG DES DEV THER. 2023 Mar 27　　WB　;　　Rat.　　 PubMed:37006723 [IF=4.292]　Wenqian Xie. et al. Seasonal expressions of ERα, ERβ, EGF, EGFR, PI3K and Akt in the scent glands of the muskrats (Ondatra zibethicus). J Steroid Biochem. 2021 Oct;213:105961　　IHC　;　　Muskrat.　　 PubMed:34391939 [IF=4.221]　Huimin Wang. et al. Chronic exposure of bisphenol-A impairs cognitive function and disrupts hippocampal insulin signaling pathway in male mice. TOXICOLOGY. 2022 Apr;472:153192　　WB　;　　Mouse.　　 PubMed:35489422 [IF=4.081]　Zhang Wenqi. et al. Alleviative Effect of Lactoferrin Interventions Against the Hepatotoxicity Induced by Titanium Dioxide Nanoparticles. BIOL TRACE ELEM RES. 2023 May;:1-19　　IHC　;　　Rat.　　 PubMed:37191759
產(chǎn)品類型	磷酸化抗體
研究領(lǐng)域	腫瘤　　細(xì)胞生物　　神經(jīng)生物學(xué)　　信號轉(zhuǎn)導(dǎo)　　細(xì)胞凋亡　　轉(zhuǎn)錄調(diào)節(jié)因子　　激酶和磷酸酶
抗體來源	Rabbit
克隆類型	Polyclonal
交叉反應(yīng)	Human,Mouse,Rat (predicted: Chicken,Dog,Pig,Cow,Rabbit,Sheep)
產(chǎn)品應(yīng)用	WB=1:500-2000, IHC-P=1:100-500, IHC-F=1:100-500, IF=1:100-500, Flow-Cyt=2ug/Test, ELISA=1:5000-10000 not yet tested in other applications. optimal dilutions/concentrations should be determined by the end user.
理論分子量	56kDa
細(xì)胞定位	細(xì)胞核　細(xì)胞漿　細(xì)胞膜
性　　　　狀	Liquid
濃　　　　度	1mg/ml
免　疫　原	KLH conjugated Synthesised phosphopeptide derived from human AKT around the phosphorylation site of Ser473:　QF(p-S)YS
亞　　　　型	IgG
純化方法	affinity purified by Protein A
緩　沖　液	0.01M TBS(pH7.4) with 1% BSA, 0.03% Proclin300 and 50% Glycerol.
保存條件	Shipped at 4℃. Store at -20 °C for one year. Avoid repeated freeze/thaw cycles.
注意事項(xiàng)	This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications.
PubMed	PubMed
產(chǎn)品介紹	This gene encodes one of the three members of the human AKT serine-threonine protein kinase family which are often referred to as protein kinase B alpha, beta, and gamma. These highly similar AKT proteins all have an N-terminal pleckstrin homology domain, a serine/threonine-specific kinase domain and a C-terminal regulatory domain. These proteins are phosphorylated by phosphoinositide 3-kinase (PI3K). AKT/PI3K forms a key component of many signalling pathways that involve the binding of membrane-bound ligands such as receptor tyrosine kinases, G-protein coupled receptors, and integrin-linked kinase. These AKT proteins therefore regulate a wide variety of cellular functions including cell proliferation, survival, metabolism, and angiogenesis in both normal and malignant cells. AKT proteins are recruited to the cell membrane by phosphatidylinositol 3,4,5-trisphosphate (PIP3) after phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) by PI3K. Subsequent phosphorylation of both threonine residue 308 and serine residue 473 is required for full activation of the AKT1 protein encoded by this gene. Phosphorylation of additional residues also occurs, for example, in response to insulin growth factor-1 and epidermal growth factor. Protein phosphatases act as negative regulators of AKT proteins by dephosphorylating AKT or PIP3. The PI3K/AKT signalling pathway is crucial for tumor cell survival. Survival factors can suppress apoptosis in a transcription-independent manner by activating AKT1 which then phosphorylates and inactivates components of the apoptotic machinery. AKT proteins also participate in the mammalian target of rapamycin (mTOR) signalling pathway which controls the assembly of the eukaryotic translation initiation factor 4F (eIF4E) complex and this pathway, in addition to responding to extracellular signals from growth factors and cytokines, is disregulated in many cancers. Mutations in this gene are associated with multiple types of cancer and excessive tissue growth including Proteus syndrome and Cowden syndrome 6, and breast, colorectal, and ovarian cancers. Multiple alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Jul 2020] Function: AKT1 is one of 3 closely related serine/threonine-protein kinases (AKT1, AKT2 and AKT3) called the AKT kinase, and which regulate many processes including metabolism, proliferation, cell survival, growth and angiogenesis. This is mediated through serine and/or threonine phosphorylation of a range of downstream substrates. Over 100 substrate candidates have been reported so far, but for most of them, no isoform specificity has been reported. AKT is responsible of the regulation of glucose uptake by mediating insulin-induced translocation of the SLC2A4/GLUT4 glucose transporter to the cell surface. Phosphorylation of PTPN1 at 'Ser-50' negatively modulates its phosphatase activity preventing dephosphorylation of the insulin receptor and the attenuation of insulin signaling. Phosphorylation of TBC1D4 triggers the binding of this effector to inhibitory 14-3-3 proteins, which is required for insulin-stimulated glucose transport. AKT regulates also the storage of glucose in the form of glycogen by phosphorylating GSK3A at 'Ser-21' and GSK3B at 'Ser-9', resulting in inhibition of its kinase activity. Phosphorylation of GSK3 isoforms by AKT is also thought to be one mechanism by which cell proliferation is driven. AKT regulates also cell survival via the phosphorylation of MAP3K5 (apoptosis signal-related kinase). Phosphorylation of 'Ser-83' decreases MAP3K5 kinase activity stimulated by oxidative stress and thereby prevents apoptosis. AKT 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. AKT is involved in the phosphorylation of members of the FOXO factors (Forkhead family of transcription factors), leading to binding of 14-3-3 proteins and cytoplasmic localization. In particular, FOXO1 is phosphorylated at 'Thr-24', 'Ser-256' and 'Ser-319'. FOXO3 and FOXO4 are phosphorylated on equivalent sites. AKT has an important role in the regulation of NF-kappa-B-dependent gene transcription and positively regulates the activity of CREB1 (cyclic AMP (cAMP)-response element binding protein). The phosphorylation of CREB1 induces the binding of accessory proteins that are necessary for the transcription of pro-survival genes such as BCL2 and MCL1. AKT phosphorylates 'Ser-454' on ATP citrate lyase (ACLY), thereby potentially regulating ACLY activity and fatty acid synthesis. Activates the 3B isoform of cyclic nucleotide phosphodiesterase (PDE3B) via phosphorylation of 'Ser-273', resulting in reduced cyclic AMP levels and inhibition of lipolysis. Phosphorylates PIKFYVE on 'Ser-318', which results in increased PI(3)P-5 activity. The Rho GTPase-activating protein DLC1 is another substrate and its phosphorylation is implicated in the regulation cell proliferation and cell growth. AKT plays a role as key modulator of the AKT-mTOR signaling pathway controlling the tempo of the process of newborn neurons integration during adult neurogenesis, including correct neuron positioning, dendritic development and synapse formation. 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). AKT mediates the antiapoptotic effects of IGF-I. Essential for the SPATA13-mediated regulation of cell migration and adhesion assembly and disassembly. May be involved in the regulation of the placental development. Phosphorylates STK4/MST1 at 'Thr-120' and 'Thr-387' leading to inhibition of its: kinase activity, nuclear translocation, autophosphorylation and ability to phosphorylate FOXO3. Phosphorylates STK3/MST2 at 'Thr-117' and 'Thr-384' leading to inhibition of its: cleavage, kinase activity, autophosphorylation at Thr-180, binding to RASSF1 and nuclear translocation. Phosphorylates SRPK2 and enhances its kinase activity towards SRSF2 and ACIN1 and promotes its nuclear translocation. Phosphorylates RAF1 at 'Ser-259' and negatively regulates its activity. Phosphorylation of BAD stimulates its pro-apoptotic activity. AKT1-specific substrates have been recently identified, including palladin (PALLD), which phosphorylation modulates cytoskeletal organization and cell motility; prohibitin (PHB), playing an important role in cell metabolism and proliferation; and CDKN1A, for which phosphorylation at 'Thr-145' induces its release from CDK2 and cytoplasmic relocalization. These recent findings indicate that the AKT1 isoform has a more specific role in cell motility and proliferation. Phosphorylates CLK2 thereby controlling cell survival to ionizing radiation. Subunit: Interacts (via the C-terminus) with CCDC88A (via its C-terminus). Interacts with GRB10; the interaction leads to GRB10 phosphorylation thus promoting YWHAE-binding. Interacts with AGAP2 (isoform 2/PIKE-A); the interaction occurs in the presence of guanine nucleotides. Interacts with AKTIP. Interacts (via PH domain) with MTCP1, TCL1A AND TCL1B. Interacts with CDKN1B; the interaction phosphorylates CDKN1B promoting 14-3-3 binding and cell-cycle progression. Interacts with MAP3K5 and TRAF6. Interacts with BAD, PPP2R5B, STK3 and STK4. Interacts (via PH domain) with SIRT1. Interacts with SRPK2 in a phosphorylation-dependent manner. Interacts with RAF1. Interacts with TRIM13; the interaction ubiquitinates AKT1 leading to its proteasomal degradation. Interacts with TNK2 and CLK2. Interacts (via the C-terminus) with THEM4 (via its C-terminus). Interacts with and phosphorylated by PDPK1. Subcellular Location: Cytoplasm. Nucleus. Cell membrane. Note=Nucleus after activation by integrin-linked protein kinase 1 (ILK1). Nuclear translocation is enhanced by interaction with TCL1A. Phosphorylation on Tyr-176 by TNK2 results in its localization to the cell membrane where it is targeted for further phosphorylations on Thr-308 and Ser-473 leading to its activation and the activated form translocates to the nucleus. Tissue Specificity: Expressed in prostate cancer and levels increase from the normal to the malignant state (at protein level). Expressed in all human cell types so far analyzed. The Tyr-176 phosphorylated form shows a significant increase in expression in breast cancers during the progressive stages i.e. normal to hyperplasia (ADH), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC) and lymph node metastatic (LNMM) stages. Post-translational modifications: O-GlcNAcylation at Thr-305 and Thr-312 inhibits activating phosphorylation at Thr-308 via disrupting the interaction between AKT1 and PDPK1. O-GlcNAcylation at Ser-473 also probably interferes with phosphorylation at this site. Phosphorylation on Thr-308, Ser-473 and Tyr-474 is required for full activity. Activated TNK2 phosphorylates it on Tyr-176 resulting in its binding to the anionic plasma membrane phospholipid PA. This phosphorylated form localizes to the cell membrane, where it is targeted by PDPK1 and PDPK2 for further phosphorylations on Thr-308 and Ser-473 leading to its activation. Ser-473 phosphorylation by mTORC2 favors Thr-308 phosphorylation by PDPK1. Ser-473 phosphorylation is enhanced by interaction with AGAP2 isoform 2 (PIKE-A). Ser-473 phosphorylation is enhanced in focal cortical dysplasias with Taylor-type balloon cells. Ser-473 phosphorylation is enhanced by signaling through activated FLT3. Dephosphorylated at Thr-308 and Ser-473 by PP2A phosphatase. The phosphorylated form of PPP2R5B is required for bridging AKT1 with PP2A phosphatase. Ubiquitinated via 'Lys-48'-linked polyubiquitination by ZNRF1, leading to its degradation by the proteasome. Ubiquitinated; undergoes both 'Lys-48'- and 'Lys-63'-linked polyubiquitination. TRAF6-induced 'Lys-63'-linked AKT1 ubiquitination is critical for phosphorylation and activation. When ubiquitinated, it translocates to the plasma membrane, where it becomes phosphorylated. When fully phosphorylated and translocated into the nucleus, undergoes 'Lys-48'-polyubiquitination catalyzed by TTC3, leading to its degradation by the proteasome. Also ubiquitinated by TRIM13 leading to its proteasomal degradation. Acetylated on Lys-14 and Lys-20 by the histone acetyltransferases EP300 and KAT2B. Acetylation results in reduced phosphorylation and inhibition of activity. Deacetylated at Lys-14 and Lys-20 by SIRT1. SIRT1-mediated deacetylation relieves the inhibition. DISEASE: Defects in AKT1 are a cause of susceptibility to breast cancer (BC) [MIM:114480]. A common malignancy originating from breast epithelial tissue. Breast neoplasms can be distinguished by their histologic pattern. Invasive ductal carcinoma is by far the most common type. Breast cancer is etiologically and genetically heterogeneous. Important genetic factors have been indicated by familial occurrence and bilateral involvement. Mutations at more than one locus can be involved in different families or even in the same case. Defects in AKT1 are associated with colorectal cancer (CRC) [MIM:114500]. Note=Genetic variations in AKT1 may play a role in susceptibility to ovarian cancer. Defects in AKT1 are a cause of Proteus syndrome (PROTEUSS) [MIM:176920]. A highly variable, severe disorder of asymmetric and disproportionate overgrowth of body parts, connective tissue nevi, epidermal nevi, dysregulated adipose tissue, and vascular malformations. Many features of Proteus syndrome overlap with other overgrowth syndromes. Similarity: Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. RAC subfamily. Contains 1 AGC-kinase C-terminal domain. Contains 1 PH domain. Contains 1 protein kinase domain. SWISS: P31749 Gene ID: 207 Database links: Entrez Gene: 207 Human Entrez Gene: 11651 Mouse Entrez Gene: 24185 Rat Omim: 164730 Human SwissProt: O57513 Chicken SwissProt: P31749 Human SwissProt: P31750 Mouse SwissProt: P47196 Rat Unigene: 525622 Human Unigene: 6645 Mouse Unigene: 11422 Rat
產(chǎn)品圖片	Sample: Heart (Mouse) Lysate at 40 ug Primary: Anti-phospho-AKT (Ser473) (bs-0876R) at 1/300 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 56 kD Observed band size: 56 kD Sample:Liver(Mouse) Lysate at 30 ug Primary: Anti-phospho-AKT(Ser473) (bs-0876R) at 1:300 dilution; Secondary: HRP conjugated Goat Anti-Rabbit IgG(bs-0295G-HRP) at 1: 5000 dilution; Predicted band size : 56kD Observed band size : 56kD Sample: U937 Cell (Human) Lysate at 30 ug Primary: Anti-phospho-AKT (Ser473) (bs-0876R) at 1/300 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 56 kD Observed band size: 56 kD Paraformaldehyde-fixed, paraffin embedded (mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-AKT (Ser473)) Polyclonal Antibody, Unconjugated (bs-0876R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining. Paraformaldehyde-fixed, paraffin embedded (rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-AKT (Ser473)) Polyclonal Antibody, Unconjugated (bs-0876R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining. Paraformaldehyde-fixed, paraffin embedded (rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-AKT (Ser473)) Polyclonal Antibody, Unconjugated (bs-0876R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining. Paraformaldehyde-fixed, paraffin embedded (rat lung); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-AKT (Ser473)) Polyclonal Antibody, Unconjugated (bs-0876R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining. Paraformaldehyde-fixed, paraffin embedded (mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-AKT (Ser473)) Polyclonal Antibody, Unconjugated (bs-0876R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining. Paraformaldehyde-fixed, paraffin embedded (rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-AKT (Ser473)) Polyclonal Antibody, Unconjugated (bs-0876R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining. Paraformaldehyde-fixed, paraffin embedded (mouse pancreas); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-AKT (Ser473)) Polyclonal Antibody, Unconjugated (bs-0876R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining. Paraformaldehyde-fixed, paraffin embedded (rat pancreas); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-AKT (Ser473)) Polyclonal Antibody, Unconjugated (bs-0876R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining. Paraformaldehyde-fixed, paraffin embedded (mouse pancreas); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-AKT (Ser473)) Polyclonal Antibody, Unconjugated (bs-0876R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining. Tissue/cell: Human breast; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min; Incubation: Anti-phospho-AKT/PKB(Ser473) Polyclonal Antibody, Unconjugated(bs-0876R) 1:600, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining Tissue/cell: Transplanted tumor in mice; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min; Incubation: Anti-phospho-AKT/PKB(Ser473) Polyclonal Antibody, Unconjugated(bs-0876R) 1:600, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining Tissue/cell: Transplanted tumor in mice; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min; Incubation: Anti-phospho-AKT/PKB(Ser473) Polyclonal Antibody, Unconjugated(bs-0876R) 1:600, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining Blank control:A549. Primary Antibody (green line): Rabbit Anti-phospho-AKT (Ser473) antibody (bs-0876R) Dilution: 2μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody : Goat anti-rabbit IgG-FITC Dilution: 1μg /test. Protocol The cells were incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed. Blank control:A549. Primary Antibody (green line): Rabbit Anti-phospho-AKT (Ser473) antibody (bs-0876R) Dilution: 2μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody : Goat anti-rabbit IgG-FITC Dilution: 1μg /test. Protocol The cells were incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.