PRODUCT CODE: ET1609-49

Recombinant Phospho FOXO3a (S253) Monoclonal Antibody (ET1609-49)

  • Recombinant

Applications

  • WB

  • IHC-P

REACTIVITY

  • Human

  • Mouse

  • Rat

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Western blot analysis of Phospho-FOXO3a(S253) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1609-49, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.<br />
 Positive control: <br />
 Lane 1: Hela cell lysate<br />
 Lane 2: PC-12 cell lysate
  • Western blot analysis of Phospho-FOXO3a(S253) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1609-49, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.<br />
 Positive control: <br />
 Lane 1: Hela cell lysate<br />
 Lane 2: PC-12 cell lysate
  • Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-Phospho-FOXO3a(S253) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1609-49, 1/50) for 30 minutes 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 human breast carcinoma tissue using anti-Phospho-FOXO3a(S253) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1609-49, 1/50) for 30 minutes 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 human pancreas tissue using anti-Phospho-FOXO3a(S253) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1609-49, 1/50) for 30 minutes 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 pancreas tissue using anti-Phospho-FOXO3a(S253) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1609-49, 1/50) for 30 minutes 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.
Western blot analysis of Phospho-FOXO3a(S253) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1609-49, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
Positive control:
Lane 1: Hela cell lysate
Lane 2: PC-12 cell lysate

Applications

  • WB

  • IHC-P

REACTIVITY

  • Human

  • Mouse

  • Rat

SPECIFICATIONS

Product Type

Recombinant Rabbit monoclonal primary

Product Name

Recombinant Phospho FOXO3a (S253) Monoclonal Antibody (ET1609-49)

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding ser253 of human foxo3a.

Host

Rabbit

Modification

Phospho

Modification Site

S253

Positive Control

Hela cell lysate, PC-12 cell lysate, human tonsil tissue, human breast carcinoma tissue, human pancreas tissue, mouse pancreas tissue.

Conjugation

Unconjugated

Clonality

Monoclonal

Clone Number

ST49-01

PROPERTIES

Form

Liquid

Storage Condition

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

Storage Buffer

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

Concentration

1 ug/ul

PURIFICATION

Protein A affinity purified.

MOLECULAR WEIGHT

71 kDa

Isotype

IgG

APPLICATION DILUTION

  • WB

  • 1:1,000-1:5,000

  • IHC-P

  • 1:50-1:200

TARGET

UNIPROT #

PROTEIN NAME

Phospho-FOXO3a(S253)

SYNONYMS

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

TISSUE SPECIFICITY

Ubiquitous.

POST-TRANSLATIONAL MODIFICATION

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, Nucleus.

FUNCTION

FKHRL1 (for forkhead in rhabdomyosarcoma) is a member of the FKHR subfamily of forkhead transcription factors. Transcriptional activation of FKHR proteins is regulated by the serine/threonine kinase Akt1, which phosphorylates FKHRL1 at threonine-32 and serine-253. Phosphorylation by Akt1 negatively regulates FKHRL1 by promoting its export from the nucleus. Phosphorylated FKHRL1 associates with 14-3-3 proteins and this complex is retained in the cytoplasm. Growth factor withdrawal stimulates FKHRL1 dephosphorylation and nuclear translocation, leading to FKHR-induced gene-specific transcriptional activation. Within the nucleus, dephospho-rylated FKHRL1 triggers apoptosis by inducing the expression of genes that are critical for cell death.

CITATIONS

  • Li, Fanni et al.

    Regulation of Akt/FoxO3a/Skp2 Axis Is Critically Involved in Berberine-Induced Cell Cycle Arrest in Hepatocellular Carcinoma Cells. | International Journal of Molecular Sciences [2018]