Product: Phospho-FOXO3A (Ser253) Antibody
Catalog: AF3020
Source: Rabbit
Application: WB, IHC, IF/ICC
Reactivity: Human, Mouse, Rat
Prediction: Pig, Bovine, Horse, Sheep, Rabbit, Dog, Chicken, Xenopus
Mol.Wt.: 97kDa; 71kD(Calculated).
Uniprot: O43524
RRID: AB_2834427

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Product Info

WB 1:500-1:2000, IHC 1:50-1:1000, IF/ICC 1:100-1:500
*The optimal dilutions should be determined by the end user.

WB: For western blot detection of denatured protein samples. IHC: For immunohistochemical detection of paraffin sections (IHC-p) or frozen sections (IHC-f) of tissue samples. IF/ICC: For immunofluorescence detection of cell samples. ELISA(peptide): For ELISA detection of antigenic peptide.

Pig(100%), Bovine(100%), Horse(100%), Sheep(100%), Rabbit(100%), Dog(100%), Chicken(100%), Xenopus(100%)
Phospho-FOXO3A (Ser253) Antibody detects endogenous levels of FOXO3A only when phosphorylated at Serine 253.
Cite Format: Affinity Biosciences Cat# AF3020, RRID:AB_2834427.
The antibody is from purified rabbit serum by affinity purification via sequential chromatography on phospho-peptide and non-phospho-peptide affinity columns.
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol. Store at -20 °C. Stable for 12 months from date of receipt.


AF6q21; AF6q21 protein; DKFZp781A0677; FKHR2; FKHRL 1; FKHRL1; FKHRL1P2; Forkhead (Drosophila) homolog (rhabdomyosarcoma) like 1; Forkhead box O3; Forkhead box O3A; Forkhead box protein O3; Forkhead box protein O3A; Forkhead Drosophila homolog of in rhabdomyosarcoma like 1; Forkhead homolog (rhabdomyosarcoma) like 1; Forkhead in rhabdomyosarcoma like 1; Forkhead in rhabdomyosarcoma-like 1; FOX O3A; FOXO2; foxo3; FOXO3_HUMAN; FOXO3A; MGC12739; MGC31925;




This gene belongs to the forkhead family of transcription factors which are characterized by a distinct forkhead domain. This gene likely functions as a trigger for apoptosis through expression of genes necessary for cell death.



Score>80(red) has high confidence and is suggested to be used for WB detection. *The prediction model is mainly based on the alignment of immunogen sequences, the results are for reference only, not as the basis of quality assurance.

Model Confidence:
High(score>80) Medium(80>score>50) Low(score<50) No confidence

PTMs - O43524 As Substrate

Site PTM Type Enzyme
S7 Phosphorylation Q16539 (MAPK14)
S12 Phosphorylation Q16539 (MAPK14)
S26 Phosphorylation
S30 Phosphorylation
T32 Phosphorylation Q9Y243 (AKT3) , Q9HBY8 (SGK2) , P67775 (PPP2CA) , P11309-2 (PIM1) , P31751 (AKT2) , P31749 (AKT1) , O00141 (SGK1)
S43 Phosphorylation
K46 Methylation
S75 Phosphorylation
S90 Phosphorylation
K149 Methylation
S161 Phosphorylation
Y162 Phosphorylation
S173 Phosphorylation
T179 Phosphorylation Q13131 (PRKAA1) , P54646 (PRKAA2)
Y184 Phosphorylation
K207 Ubiquitination
S209 Phosphorylation Q13043 (STK4)
S215 Phosphorylation Q13043 (STK4) , Q8IW41 (MAPKAPK5)
K230 Methylation
S231 Phosphorylation Q13043 (STK4)
S232 Phosphorylation Q13043 (STK4)
K242 Acetylation
S243 Phosphorylation Q13043 (STK4)
K245 Acetylation
S253 Phosphorylation Q9Y243 (AKT3) , O00141 (SGK1) , Q96BR1 (SGK3) , Q9HBY8 (SGK2) , P11309-2 (PIM1) , P31749 (AKT1) , P31751 (AKT2)
K259 Acetylation
K262 Methylation
K271 Acetylation
K271 Methylation
S280 Phosphorylation
S284 Phosphorylation
S286 Phosphorylation
S289 Phosphorylation
K290 Acetylation
K290 Methylation
S294 Phosphorylation P28482 (MAPK1) , Q16539 (MAPK14) , P27361 (MAPK3)
T296 Phosphorylation
S297 Phosphorylation
S299 Phosphorylation
T307 Phosphorylation
S311 Phosphorylation
S315 Phosphorylation P31749 (AKT1) , O00141 (SGK1) , P31751 (AKT2) , Q9Y243 (AKT3)
S318 Phosphorylation P48729 (CSNK1A1)
T319 Phosphorylation
S321 Phosphorylation P48729 (CSNK1A1)
S325 Phosphorylation
S330 Phosphorylation Q13627 (DYRK1A)
S344 Phosphorylation Q16539 (MAPK14) , P27361 (MAPK3) , P28482 (MAPK1)
S355 Phosphorylation
T395 Phosphorylation
S399 Phosphorylation P54646 (PRKAA2) , Q13131 (PRKAA1)
S402 Phosphorylation
S413 Phosphorylation P54646 (PRKAA2) , Q13131 (PRKAA1)
K419 Methylation
S421 Phosphorylation
S425 Phosphorylation P27361 (MAPK3) , P28482 (MAPK1) , Q16539 (MAPK14)
T427 Phosphorylation
S439 Phosphorylation
S551 Phosphorylation
S553 Phosphorylation
S555 Phosphorylation Q13131 (PRKAA1) , P54646 (PRKAA2)
K569 Acetylation
S574 Phosphorylation P45983 (MAPK8)
S588 Phosphorylation P54646 (PRKAA2) , Q13131 (PRKAA1)
S626 Phosphorylation P54646 (PRKAA2) , Q13131 (PRKAA1)
S644 Phosphorylation Q14164 (IKBKE) , O15111 (CHUK) , O14920 (IKBKB)

Research Backgrounds


Transcriptional activator that recognizes and binds to the DNA sequence 5'-[AG]TAAA[TC]A-3' and regulates different processes, such as apoptosis and autophagy. Acts as a positive regulator of autophagy in skeletal muscle: in starved cells, enters the nucleus following dephosphorylation and binds the promoters of autophagy genes, such as GABARAP1L, MAP1LC3B and ATG12, thereby activating their expression, resulting in proteolysis of skeletal muscle proteins (By similarity). Triggers apoptosis in the absence of survival factors, including neuronal cell death upon oxidative stress. Participates in post-transcriptional regulation of MYC: following phosphorylation by MAPKAPK5, promotes induction of miR-34b and miR-34c expression, 2 post-transcriptional regulators of MYC that bind to the 3'UTR of MYC transcript and prevent its translation. In response to metabolic stress, translocates into the mitochondria where it promotes mtDNA transcription.


In the presence of survival factors such as IGF-1, phosphorylated on Thr-32 and Ser-253 by AKT1/PKB. This phosphorylated form then interacts with 14-3-3 proteins and is retained in the cytoplasm. Survival factor withdrawal induces dephosphorylation and promotes translocation to the nucleus where the dephosphorylated protein induces transcription of target genes and triggers apoptosis. Although AKT1/PKB doesn't appear to phosphorylate Ser-315 directly, it may activate other kinases that trigger phosphorylation at this residue. Phosphorylated by STK4/MST1 on Ser-209 upon oxidative stress, which leads to dissociation from YWHAB/14-3-3-beta and nuclear translocation. Phosphorylated by PIM1. Phosphorylation by AMPK leads to the activation of transcriptional activity without affecting subcellular localization. In response to metabolic stress, phosphorylated by AMPK on Ser-30 which mediates FOXO3 mitochondrial translocation. Phosphorylation by MAPKAPK5 promotes nuclear localization and DNA-binding, leading to induction of miR-34b and miR-34c expression, 2 post-transcriptional regulators of MYC that bind to the 3'UTR of MYC transcript and prevent its translation. Phosphorylated by CHUK/IKKA and IKBKB/IKKB. TNF-induced inactivation of FOXO3 requires its phosphorylation at Ser-644 by IKBKB/IKKB which promotes FOXO3 retention in the cytoplasm, polyubiquitination and ubiquitin-mediated proteasomal degradation. May be dephosphorylated by calcineurin A on Ser-299 which abolishes FOXO3 transcriptional activity (By similarity). In cancer cells, ERK mediated-phosphorylation of Ser-12 is required for mitochondrial translocation of FOXO3 in response to metabolic stress or chemotherapeutic agents.

Deacetylation by SIRT1 or SIRT2 stimulates interaction of FOXO3 with SKP2 and facilitates SCF(SKP2)-mediated FOXO3 ubiquitination and proteasomal degradation. Deacetylation by SIRT2 stimulates FOXO3-mediated transcriptional activity in response to oxidative stress (By similarity). Deacetylated by SIRT3. Deacetylation by SIRT3 stimulates FOXO3-mediated mtDNA transcriptional activity in response to metabolic stress.

Heavily methylated by SET9 which decreases stability, while moderately increasing transcriptional activity. The main methylation site is Lys-271. Methylation doesn't affect subcellular location.

Polyubiquitinated. Ubiquitinated by a SCF complex containing SKP2, leading to proteasomal degradation.

The N-terminus is cleaved following import into the mitochondrion.

Subcellular Location:

Cytoplasm>Cytosol. Nucleus. Mitochondrion matrix. Mitochondrion outer membrane>Peripheral membrane protein>Cytoplasmic side.
Note: Retention in the cytoplasm contributes to its inactivation (PubMed:10102273, PubMed:15084260, PubMed:16751106). Translocates to the nucleus upon oxidative stress and in the absence of survival factors (PubMed:10102273, PubMed:16751106). Translocates from the cytosol to the nucleus following dephosphorylation in response to autophagy-inducing stimuli (By similarity). Translocates in a AMPK-dependent manner into the mitochondrion in response to metabolic stress (PubMed:23283301, PubMed:29445193).

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Tissue Specificity:


Subunit Structure:

Upon metabolic stress, forms a complex composed of FOXO3, SIRT3 and mitochondrial RNA polymerase POLRMT; the complex is recruited to mtDNA in a SIRT3-dependent manner. Also forms a complex composed of FOXO3, SIRT3, TFAM and POLRMT. Interacts with SIRT2; the interaction occurs independently of SIRT2 deacetylase activity (By similarity). Interacts with YWHAB/14-3-3-beta and YWHAZ/14-3-3-zeta, which are required for cytosolic sequestration. Upon oxidative stress, interacts with STK4/MST1, which disrupts interaction with YWHAB/14-3-3-beta and leads to nuclear translocation. Interacts with PIM1. Interacts with DDIT3/CHOP. Interacts (deacetylated form) with SKP2. Interacts with CHUK and IKBKB. Interacts with CAMK2A, CAMK2B and calcineurin A (By similarity). Interacts FOXO3; this interaction represses FOXO3 transactivation.

Research Fields

· Cellular Processes > Cell growth and death > Cellular senescence.   (View pathway)

· Environmental Information Processing > Signal transduction > FoxO signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > PI3K-Akt signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > AMPK signaling pathway.   (View pathway)

· Human Diseases > Drug resistance: Antineoplastic > EGFR tyrosine kinase inhibitor resistance.

· Human Diseases > Cancers: Specific types > Endometrial cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Non-small cell lung cancer.   (View pathway)

· Organismal Systems > Immune system > Chemokine signaling pathway.   (View pathway)

· Organismal Systems > Aging > Longevity regulating pathway.   (View pathway)

· Organismal Systems > Aging > Longevity regulating pathway - multiple species.   (View pathway)

· Organismal Systems > Nervous system > Neurotrophin signaling pathway.   (View pathway)

· Organismal Systems > Endocrine system > Prolactin signaling pathway.   (View pathway)


1). Hu R et al. Calycosin inhibited autophagy and oxidative stress in chronic kidney disease skeletal muscle atrophy by regulating AMPK/SKP2/CARM1 signalling pathway. J Cell Mol Med 2020 Oct;24(19):11084-11099. (PubMed: 32910538) [IF=5.295]

Application: WB    Species: mouse    Sample: C2C12 cells

FIGURE 8| Effect of calycosin on the expression of proteins associated with the AMPK/SKP2/CARM1 signalling pathway in TNF-αinduced C2C12 cells in vitro. A, Representative images of H3R17me2a in C2C12 cells treated with TNF-α or calycosin. B-C, Representative immunoblot of key proteins associated with the AMPK/SKP2/CARM1 signalling pathway in C2C12 cells treated with TNF-α or calycosin.

2). Liu L et al. Formononetin ameliorates muscle atrophy by regulating myostatin‐mediated PI3K/Akt/FoxO3a pathway and satellite cell function in chronic kidney disease. J Cell Mol Med 2021 Jan 6. (PubMed: 33405354) [IF=5.295]

Application: WB    Species: Mouse    Sample: C2C12 myoblasts

FIGURE 5 FMN inhibits myostatin-mediated dephosphorylation on the PI3K/Akt/FoxO3a signalling pathway in the muscle of CKD rats and C2C12 myotubes. (A) Protein levels of p-PI3K, PI3K, p-Akt, Akt, p-FoxO3a and FoxO3a in gastrocnemius muscle analysed using Western blotting (n = 3/group). (B) Protein levels of MAFbx and MuRF-1 in gastrocnemius muscles analysed using Western blotting (n = 3/ group). (C) C2C12 myotubes were treated with FMN (50 μmol/L) in the presence or absence of TNF-α (40 ng/mL) for 48 h following 24 h of incubation with si-myostatin or si-NC. The myotubes were divided into four groups: si-NC, si-NC + TNF-α, si-NC + TNF-α + FMN (50 μmol/L) and si-myostatin + TNF-α. Protein levels of p-PI3K, PI3K, p-Akt, Akt, p-FoxO3a and FoxO3a in C2C12 myotubes (n = 3/ group). (D) Protein levels of MAFbx and MuRF-1 in C2C12 myotubes (n = 3/group). (E) Myostatin OE transfection was used to overexpress myostatin in C2C12 myotubes, and they were incubated with FMN (50 μmol/L) and TNF-α for another 48 h. The myotubes were divided into four groups: vector NC, vector NC + TNF-α, vector NC + TNF-α + FMN (50 μmol/L) and myostatin OE + TNF-α + FMN (50 μmol/L). The protein levels of p-PI3K, PI3K, p-Akt, Akt, p-FoxO3a and FoxO3a in the myotubes were analysed using Western blotting. (F) Protein levels of MAFbx and MuRF1 in C2C12 myotubes analysed by Western blotting (n = 3/group). *P < .05, **P < .01

3). Li Z et al. Berberine inhibits RA-FLS cell proliferation and adhesion by regulating RAS/MAPK/FOXO/HIF-1 signal pathway in the treatment of rheumatoid arthritis. Bone Joint Res 2023 Feb;12(2):91-102. (PubMed: 36718649) [IF=4.410]

4). Han Y et al. MicroRNA‑21‑5p acts via the PTEN/Akt/FOXO3a signaling pathway to prevent cardiomyocyte injury caused by high glucose/high fat conditions. Exp Ther Med 2022 Mar;23(3):230. (PubMed: 35222707) [IF=2.751]

Application: WB    Species: Rat    Sample: H9c2 cells

Figure 2 Effects of high levels of glucose and fat on expression of apoptosis-related and PTEN/Akt/FOXO3a proteins in H9c2 cells. **P<0.01 compared with the control group (n=6 in each group). HG-HF, high glucose-high fat; p-, phosphorylated.

5). MicroRNA‑21‑5p acts via the PTEN/Akt/FOXO3a signaling pathway to prevent cardiomyocyte injury caused by high glucose/high fat conditions.

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