Phospho-AKT (S473) Recombinant Rabbit Monoclonal Antibody [SY28-05]

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Catalog# ET1607-73

Phospho-AKT (S473) Recombinant Rabbit Monoclonal Antibody [SY28-05]

Application

WB
IF-Cell
IF-Tissue
IHC-P
IHC-Fr

Reactivity

Human
Mouse
Rat
Dog

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

Phospho-AKT (S473) Recombinant Rabbit Monoclonal Antibody [SY28-05]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding Ser473 of human AKT1.

Species Reactivity

Human, Mouse, Rat, Dog

Validated Applications

WB, IF-Cell, IF-Tissue, IHC-P, IHC-Fr

Molecular Weight

Predicted band size: 56 kDa

Positive Control

MCF7 treated with 100nM Calyculin A for 30 minutes cell lysate, NIH/3T3 treated with 100ng/mL PDGF for 1 hour cell lysate, C6 treated with 100nM Calyculin A for 30 minutes cell lysate, HEK-293 cell lysate, HeLa cells treated with 100nM Calyculin A for 30 minutes, NIH/3T3 cells treated with 100ng/mL PDGF for 1 hour, C6 cells treated with 100nM Calyculin A for 30 minutes, human breast cancer tissue, mouse lung tissue, rat brain tissue, mouse hippocampus tissue, mouse cerebral cortex tissue, mouse brain tissue.

Conjugation

unconjugated

Clone Number

SY28-05

RRID

AB_2940863

Product Features

Form

Liquid

Concentration

1ug/ul

Storage Instructions

Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

WB
1:5,000-1:10,000

IF-Cell
1:100-1:1,000

IHC-P
1:200-1:1,000

IF-Tissue
1:200-1:500

IHC-Fr
1:100

Applications in Publications

WB	See 68 publications below
IF	See 3 publications below
CO-IP	See 1 publications below
IHC	See 1 publications below
IF;WB	See 1 publications below

Species in Publications

Human	See 35 publications below
Mouse	See 22 publications below
Rat	See 5 publications below
rat	See 2 publications below
Hen	See 2 publications below
Zebrafish	See 1 publications below
Goat	See 1 publications below
Human hepatocellular carcinoma cell	See 1 publications below
Fungi	See 1 publications below
Rabbit	See 1 publications below
Human	See 1 publications below
Dog	See 1 publications below

Target

Function

RAC(Rho family)-alpha serine/threonine-protein kinase is an enzyme that in humans is encoded by the AKT1 gene. This enzyme belongs to the AKT subfamily of serine/threonine kinases that contain SH2 (Src homology 2-like) protein domains. It is commonly referred to as PKB, or by both names as "Akt/PKB". The serine-threonine protein kinase AKT1 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.

Background References

1. Lee DS et al. P2 × 7 Receptor Inhibits Astroglial Autophagy via Regulating FAK- and PHLPP1/2-Mediated AKT-S473 Phosphorylation Following Kainic Acid-Induced Seizures. Int J Mol Sci. 2020 Sep

2. Cai Q et al. MAPK6-AKT signaling promotes tumor growth and resistance to mTOR kinase blockade. Sci Adv. 2021 Nov

Sequence Similarity

Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. RAC subfamily.

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 Modification

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. Phosphorylated at Thr-308 and Ser-473 by IKBKE and TBK1. 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 (By similarity). Ser-473 is dephosphorylated by PHLPP. Dephosphorylated at Thr-308 and Ser-473 by PP2A phosphatase. The phosphorylated form of PPP2R5B is required for bridging AKT1 with PP2A phosphatase. Ser-473 is dephosphorylated by CPPED1, leading to termination of signaling.; Ubiquitinated via 'Lys-48'-linked polyubiquitination by ZNRF1, leading to its degradation by the proteasome (By similarity). 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. Phosphorylated, undergoes 'Lys-48'-linked polyubiquitination preferentially at Lys-284 catalyzed by MUL1, 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.

Subcellular Location

Cytoplasm, Nucleus, Cell membrane.

UNIPROT

SwissProt: P31749 Human

SwissProt: P31751 Human

SwissProt: Q9Y243 Human

SwissProt: P31750 Mouse

SwissProt: P47196 Rat

Synonyms

AKT 1 antibody

AKT antibody

AKT1 antibody

AKT1_HUMAN antibody

MGC99656 antibody

PKB antibody

PKB-ALPHA antibody

PRKBA antibody

Protein Kinase B Alpha antibody

Protein kinase B antibody

Expand

Images

☑ Cell treatment (CT)

Western blot analysis of Phospho-AKT (S473) on different lysates with Rabbit anti-Phospho-AKT (S473) antibody (ET1607-73) at 1/5,000 dilution.

Lane 1: MCF7 cell lysate
Lane 2: MCF7 treated with 100nM Calyculin A for 30 minutes cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: NIH/3T3 treated with 100ng/mL PDGF for 1 hour cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 56 kDa
Observed band size: 56 kDa

Exposure time: 53 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1607-73) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
☑ Cell treatment (CT)

Western blot analysis of Phospho-AKT (S473) on different lysates with Rabbit anti-Phospho-AKT (S473) antibody (ET1607-73) at 1/5,000 dilution.

Lane 1: C6 cell lysate
Lane 2: C6 treated with 100nM Calyculin A for 30 minutes cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 56 kDa
Observed band size: 56 kDa

Exposure time: 3 minutes; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1607-73) at 1/5,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
☑ Cell treatment (CT)

Western blot analysis of Phospho-AKT (S473) on different lysates with Rabbit anti-Phospho-AKT (S473) antibody (ET1607-73) at 1/5,000 dilution.

Lane 1: HEK-293 cell lysate
Lane 2: HEK-293 treated with 50μM LY294002 for 6 hours cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 56 kDa
Observed band size: 56 kDa

Exposure time: 50 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1607-73) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/200,000 dilution was used for 1 hour at room temperature.
☑ Cell treatment (CT)

Immunocytochemistry analysis of HeLa cells treated with or without 100nM Calyculin A for 30 minutes labeling Phospho-AKT (S473) with Rabbit anti-Phospho-AKT (S473) antibody (ET1607-73) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-AKT (S473) antibody (ET1607-73) at 1/200 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
☑ Cell treatment (CT)

Immunocytochemistry analysis of NIH/3T3 cells treated with or without 100ng/mL PDGF for 1 hour labeling Phospho-AKT (S473) with Rabbit anti-Phospho-AKT (S473) antibody (ET1607-73) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-AKT (S473) antibody (ET1607-73) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
☑ Cell treatment (CT)

Immunocytochemistry analysis of C6 cells treated with or without 100nM Calyculin A for 30 minutes labeling Phospho-AKT (S473) with Rabbit anti-Phospho-AKT (S473) antibody (ET1607-73) at 1/1,000 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-AKT (S473) antibody (ET1607-73) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
Immunohistochemical analysis of paraffin-embedded human breast cancer tissue with Rabbit anti-Phospho-AKT (S473) antibody (ET1607-73) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-73) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded mouse lung tissue with Rabbit anti-Phospho-AKT (S473) antibody (ET1607-73) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-73) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-AKT (S473) antibody (ET1607-73) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1607-73) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Application: IHC-Fr

Species: Mouse

Site: Cerebral cortex

Sample: Frozen section

Antibody concentration: 1/200

Antigen retrieval: Not required
Application: IF-tissue

Species: Human

Site: Breast cancer

Sample: Paraffin-embedded section

Antibody concentration: 1/200
Application: IF-tissue

Species: Mouse

Site: Lung

Sample: Paraffin-embedded section

Antibody concentration: 1/200
Application: IF-tissue

Species: Mouse

Site: Cerebral cortex

Sample: Paraffin-embedded section

Antibody concentration: 1/200