Antibody Panel to AKT Protein Family - Product Review 10

Introduction

Putative human homologs of the protooncogene v-akt of the acutely transforming retrovirus AKT8 have been cloned. These protein-serine/threonine kinase proteins have a catalytic domain closely related to both PKA and PKC and have been designated rac (related to A and C kinases), PKB (Protein kinase B) or AKT.

RAC protein kinase family members feature pleckstrin homology (PH) domain at the amino terminus and a protein-serine/ threonine kinase catalytic domain at the carboxy terminus. The Amino terminal domain (referred to as AH-Akt Homology domain) spans from 1-148 amino acids and contains the PH domain, a region found in diverse group of signaling proteins. The PH domain (amino acids 1-106) has been implicated in interactions with other proteins such as G-protein bg subunits, as well as phosphoinositides. The kinase domain is located between residues 148 to 411. These enzymes are activated by diverse ligands such as PGDF, EGF and basic FG in NIH 3T3, Rat-1 or Swiss-3T3 cells.

AKT1 or Protein kinase B alpha is a component of the PI-3 kinase pathway and is activated by phosphorylation at Ser 473 and Thr 308. AKT1 is a key regulator of many signal transduction pathways. AKT1 Exhibits tight control over cell proliferation and cell viability. Overexpression or inappropriate activation of AKT1 is noted in many types of cancer. AKT1 mediates many of the downstream events of PI 3-kinase (a lipid kinase activated by growth factors, cytokines and insulin).

PI 3-kinase recruits AKT1 to the membrane, where it is activated by PDK1 phosphorylation. Once phosphorylated, AKT1 dissociates from the membrane and phosphorylates targets in the cytoplasm and the cell nucleus. AKT1 has two main roles: (i) inhibition of apoptosis; (ii) promotion of proliferation.

AKT2 or Protein kinase B beta is an isoform of the phosphoinositidedependent serine-threonine protein kinase AKT and is enriched in insulin-responsive tissues and has been implicated in the metabolic actions of the hormone. AKT2 is a putative oncogene encoding a protein belonging to a subfamily of serine/threonine kinases containing SH2-like (Src homology 2-like) domains. Furthermore, AKT2 was shown to be amplified and overexpressed in 2 of 8 ovarian carcinoma cell lines and 2 of 15 primary ovarian tumors. Overexpression of AKT2 contributes to the malignant phenotype of a subset of human ductal pancreatic cancers. AKT2 is a general protein kinase capable of phophorylating several known proteins. AKT2 mediates many of the downstream events of PI 3-kinase (a lipid kinase activated by growth factors, cytokines and insulin). PI 3-kinase recruits AKT2 to the membrane, where it is activated by PDK1 phosphorylation. Once phosphorylated, AKT2 dissociates from the membrane and phosphorylates targets in the cytoplasm and the cell nucleus. AKT2 has two main roles: (i) inhibition of apoptosis; (ii) promotion of proliferation.

AKT3 or Protein Kinase B gamma (RAC-PK-g, rat 454-aa, mouse/human 479-aa) is highly related to other members of RAC protein kinase family. It is abundantly expressed in kidney, lung, and brain, but weakly in heart, testis, and liver. It is cytoplasmic and membrane associated after cell stimulation leading to its translocation. AKT3 is alternatively spliced to Gamma-1 and -2 isoform. It is phosphorylated on Thr and Ser, which are required for full activity. AKT3 is involved in regulation of cellular growth.

Antibody Tools for Detection of AKT Family Members

Monoclonal and polyclonal antibodies specific for AKT1, AKT2 and AKT3 and for the phosphorylated forms are available at Acris Antibodies. For more informations like host, isotype, species cross-reactivity etc. see table 1 at the end of this review.

Picture Gallery

Figure 1. Immunofluorescence microscopy. Rabbit anti-AKT was used at a 1:80 dilution to stain cultured neonatal rat cardiomyocytes that express a nuclear-targeted AKT construct. AntiAKT staining appears green. Actin filaments are labeled red using a Texas-red™ conjugated phalloidin.

Figure 1. Immunofluorescence microscopy. Rabbit anti-AKT was used at a 1:80 dilution to stain cultured neonatal rat cardiomyocytes that express a nuclear-targeted AKT construct. AntiAKT staining appears green. Actin filaments are labeled red using a Texas-red™ conjugated phalloidin.

Figure 2. Immunoblotting. Rabbit anti-AKT was used at a 1:500 dilution to detect AKT by Western blot. Approximately 20 ug of an NIH/3T3 whole cell lysate was loaded on a 10% NuPage SDS-PAGE gel using MOPS buffer. After washing, a 1:10,000 dilution of HRP conjugated Gt-a-Rabbit IgG (R1454HRP) preceded color development using Pierce Chemical's SuperSignal™ substrate.

Figure 2. Immunoblotting. Rabbit anti-AKT was used at a 1:500 dilution to detect AKT by Western blot. Approximately 20 ug of an NIH/3T3 whole cell lysate was loaded on a 10% NuPage SDS-PAGE gel using MOPS buffer. After washing, a 1:10,000 dilution of HRP conjugated Gt-a-Rabbit IgG (R1454HRP) preceded color development using Pierce Chemical's SuperSignal™ substrate.

Figure 1. Immunofluorescence microscopy. Rabbit anti-AKT was used at a 1:80 dilution to stain cultured neonatal rat cardiomyocytes that express a nuclear-targeted AKT construct. AntiAKT staining appears green. Actin filaments are labeled red using a Texas-red™ conjugated phalloidin.
Figure 3 and 4: Immunohistochemistry. Rabbit anti-AKT was used at a 1:1,000 dilution to detect AKT by immunohistochemistry. In panel (A) normal colon tissue is stained. Panel (B) shows tumor tissue. Tissue was formalin-fixed and paraffin embedded.

Figure 5. Immunohistochemistry. Rabbit anti-AKT pS473 cat# R1465 was used at a 1:100 dilution to detect AKT by immunohistochemistry in human breast tumor tissue. The staining is much stronger than the weak basal level of phosphorylation in normal breast. Rabbit anti-AKT pS473 antibody was used with no pretreatment of tissue. Signal was developed using Dako's Techmate streptavidinbiotin reagents. Tissue was formalin-fixed and paraffin embedded
Figure 5. Immunohistochemistry. Rabbit anti-AKT pS473 cat# R1465 was used at a 1:100 dilution to detect AKT by immunohistochemistry in human breast tumor tissue. The staining is much stronger than the weak basal level of phosphorylation in normal breast. Rabbit anti-AKT pS473 antibody was used with no pretreatment of tissue. Signal was developed using Dako's Techmate streptavidinbiotin reagents. Tissue was formalin-fixed and paraffin embedded.
Figure 6. Immunofluorescence Microscopy. Confocal image using Rabbit anti-AKT pS473 cat# R1465 at a 1:40 dilution to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus expressing with wild-type AKT in conjunction with a texas-red conjugated phalloidin (red) to label filamentous actin.
Figure 6. Immunofluorescence Microscopy. Confocal image using Rabbit anti-AKT pS473 cat# R1465 at a 1:40 dilution to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus expressing with wild-type AKT in conjunction with a texas-red conjugated phalloidin (red) to label filamentous actin.
Figure 7. Immunoblotting of R1467. Antibody to AKT2 detects both unphosphorylated and phosphorylated AKT2. As before approximately 20mg/lane of crude HEK293 lysate was loaded for SDS-PAGE, separated and then transferred to nitrocellulose. The right lane contains lysate pretreated for 15min with 25uM LY294002 which affects the phosphorylation of endogenous AKT2 but has no effect on phosphorylation of the myristoylated construct. This antibody clearly detects both the phosphorylated (top arrow) and the non-phosphorylated (bottom arrow) forms of endogenous AKT2.
Figure 7. Immunoblotting of R1467. Antibody to AKT2 detects both unphosphorylated and phosphorylated AKT2. As before approximately 20mg/lane of crude HEK293 lysate was loaded for SDS-PAGE, separated and then transferred to nitrocellulose. The right lane contains lysate pretreated for 15min with 25uM LY294002 which affects the phosphorylation of endogenous AKT2 but has no effect on phosphorylation of the myristoylated construct. This antibody clearly detects both the phosphorylated (top arrow) and the non-phosphorylated (bottom arrow) forms of endogenous AKT2.
Figure 8. Immunoblotting of R1467. Affinity Purified antibody to AKT2 was used at a 1:1,000 dilution to detect AKT2 by Western blot. A lysate from a stable HEK293 cell line expressing an inducible, myristoylated form of Akt2 (MyrAkt2-ER) was loaded for SDSPAGE,separated and then transferred to nitrocellulose. The blot was reacted with primary antibody for 1h at room temperature. In response to 4OHT (tamoxifen), the Akt2 is recruited to the plasma membrane via its myristoylation sequence and becomes phosphorylated and activated. The blot (right panel) shows 0 m, 15 m, and 30 m of 4OHT treatment. This treatment has no effect on endogenous AKT2, but causes a band shift upwards in the MyrAKT2. Endogenous AKT2 runs at about 60kDa whereas the myristoylatedconstruct runs at around 110kDa.
Figure 8. Immunoblotting of R1467. Affinity Purified antibody to AKT2 was used at a 1:1,000 dilution to detect AKT2 by Western blot. A lysate from a stable HEK293 cell line expressing an inducible, myristoylated form of Akt2 (MyrAkt2-ER) was loaded for SDSPAGE,separated and then transferred to nitrocellulose. The blot was reacted with primary antibody for 1h at room temperature. In response to 4OHT (tamoxifen), the Akt2 is recruited to the plasma membrane via its myristoylation sequence and becomes phosphorylated and activated. The blot (right panel) shows 0 m, 15 m, and 30 m of 4OHT treatment. This treatment has no effect on endogenous AKT2, but causes a band shift upwards in the MyrAKT2. Endogenous AKT2 runs at about 60kDa whereas the myristoylatedconstruct runs at around 110kDa.
Figure 9. Western blot analysis for AKT1 using SM7001P at 2 µg/ml dilution against 10 µg/lane of untreated (lane 1) and PDGF treated (lane 2) NIH-3T3 lysate.
Figure 9. Western blot analysis for AKT1 using SM7001P at 2 µg/ml dilution against 10 µg/lane of untreated (lane 1) and PDGF treated (lane 2) NIH-3T3 lysate.
Figure 10. Western blot analysis of Akt2 using SM7002P at 1:1000 against 30 µg of human placenta lysate.
Figure 10. Western blot analysis of Akt2 using SM7002P at 1:1000 against 30 µg of human placenta lysate.

Available products

Anti- AKT, AKT1 (PKB alpha, RAC), AKT2 (PKB beta), AKT3 (PKB gamma, PKBG) antibody

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Antibodies

Catalog No. Host Clone/Iso. Pres. React. Applications  
AM00107PU-N

AKT1 / PKB non pSer473 (incl. pos. control) antibody

Detection of endogenous PKB:
Whole cell lysates of serum starved tumor cells (20.000 cells per lane) were applied to SDS-PAGE and transferred to a PVDF membrane. The immunoblot was probed with mab 11A11 (1 μg/ ml) for 1h at RT and developed by ECL (exp. time: 30 sec).
lane 1: Hela; lane 2: HepG2; lane 3: HEK293; lane 4: SH-SY5Y;
lane 5: MDCK; lane 6: PC12; lane 7: CMT; lane 8: Neuro 2A;
lane 9: 3T3 Mouse 11A11 Purified Can, Hu, Ms, Rt E, WB
0.1 mg / €340.00
  IgG1   Acris Antibodies GmbH
 

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