The immunogen was raised against Human AKT1 phosphorylated at serine 473, however due to the high degree of similarity between AKT1, AKT2 and AKT3 the antibody is also predicted to react with all these isoforms.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use a concentration of 1 µg/ml. Detects a band of approximately 57 kDa (predicted molecular weight: 57 kDa).
Plays a role as a 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 (By similarity). 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. The activated form can suppress FoxO gene transcription and promote cell cycle progression. Essential for the SPATA13-mediated regulation of cell migration and adhesion assembly and disassembly.
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.
Involvement in 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]. Defects in AKT1 are associated with susceptibility to ovarian cancer [MIM:604370]; also called susceptibility to familial breast-ovarian cancer type 1 (BROVCA1).
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.
Binding of the PH domain to the phosphatidylinositol 3-kinase alpha (PI(3)K) results in its targeting to the plasma membrane. The PH domain mediates interaction with TNK2 and Tyr-176 is also essential for this interaction. The AGC-kinase C-terminal mediates interaction with THEM4.
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. 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.
Cytoplasm. Nucleus. Cell membrane. 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.
Western blot - Anti-AKT1 (phospho S473) antibody (ab18206)
All lanes : Anti-AKT1 (phospho S473) antibody (ab18206) at 1 µg/ml
Lane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate Lane 2 : NIH 3T3 treated with Vanadate and PDGF Whole Cell Lysate Lane 3 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate with Human pan-AKT (phospho S473) peptide (ab18622) at 1 µg/ml Lane 4 : NIH 3T3 treated with Vanadate and PDGF Whole Cell Lysate with Human pan-AKT (phospho S473) peptide (ab18622) at 1 µg/ml Lane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate with Human AKT1 (unmodified ) peptide (ab24649) at 1 µg/ml Lane 6 : NIH 3T3 treated with Vanadate and PDGF Whole Cell Lysate with Human AKT1 (unmodified ) peptide (ab24649) at 1 µg/ml
Lysates/proteins at 20 µg per lane.
Secondary All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
We are unsure of the identity of the higher bands detected at ~78 and 130 kDa. We believe this could be cross reaction with other phospho proteins, however the target band at 57 kDa is detected only in PDGF treated lysate and this signal is quenched by addition of the immunising peptide suggesting a specific interaction.
IHC image of AKT1 (phospho S473) staining in mouse lung formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab18206, 5µg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.