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Phospho-FOXO3a (S253) Recombinant Rabbit Monoclonal Antibody [ST49-01]

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Specification

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Catalog# ET1609-49

Phospho-FOXO3a (S253) Recombinant Rabbit Monoclonal Antibody [ST49-01]

Application

  • WB

  • IHC-P

  • IF-Cell

  • FC

Reactivity

  • Human

  • Mouse

  • Rat

BSA and Azide free
Conjugation

  • unconjugated

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

Overview

Product Name

Phospho-FOXO3a (S253) Recombinant Rabbit Monoclonal Antibody [ST49-01]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic Human Metabotropic Glutamate Receptor 5 aa 1150 to the C-terminus aa 238-282 / 673.

Species Reactivity

Human, Mouse, Rat

Validated Applications

WB, IHC-P, IF-Cell, FC

Molecular Weight

Predicted band size: 71 kDa

Positive Control

Hela cell lysate, PC-12 cell lysate, THP-1 cell lysate, K562 cell lysate, human tonsil tissue, human breast carcinoma tissue, human pancreas tissue, mouse pancreas tissue, 293T whole cell lysate, 293T treated with 50 uM LY294002 and 1 uM Wortmannin for 16 hours whole cell lysate.

Conjugation

unconjugated

Clone Number

ST49-01

RRID

AB_3069859

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). Store at -20℃ long term.

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

  • 1:1,000-1:5,000

  • IHC-P

  • 1:50-1:200

  • IF-Cell

  • 1:100

  • FC

  • 1:1,000

Target

Function

FOXO3 belongs to the O subclass of the forkhead family of transcription factors which are characterized by a distinct fork head DNA-binding domain. There are three other FoxO family members in humans, FOXO1, FOXO4 and FOXO6. These transcription factors share the ability to be inhibited and translocated out of the nucleus on phosphorylation by proteins such as Akt/PKB in the PI3K signaling pathway (aside from FOXO6, which may be constitutively nuclear). Other post-translational modifications including acetylation and methylation are seen and can result in increased or altered FOXO3a activity. The use of FOXO3a knockout mice has revealed a diverse range of functions in both health and disease, namely infertility, lymphoproliferation, adenoma, organ inflammation, metabolism etc.; yet despite the purported importance of FOXO transcription factors in aging, FOXO3A knockout mice do not show an obvious shortening of lifespan or accelerated aging.

Background References

1. Guillen-Ahlers H et al. Fas/CD95 Deficiency in Apc Mice Increases Intestinal Tumor Burden. PLoS One 5:e9070 (2010).

2. Mojsilovic-Petrovic J et al. FOXO3a is broadly neuroprotective in vitro and in vivo against insults implicated in motor neuron diseases. J Neurosci 29:8236-47 (2009).

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, Membrane, Mitochondrion, Mitochondrion outer membrane, Nucleus.

UNIPROT

SwissProt: O43524 Human

SwissProt: Q9WVH4 Mouse

SwissProt: D3ZBQ1 Rat

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

Expand

Images

  • Western blot analysis of Phospho-FOXO3a (S253) on different lysates with Rabbit anti-Phospho-FOXO3a (S253) antibody (<a href="/products/ET1609-49" style="font-weight: bold;text-decoration: underline;">ET1609-49</a>) at 1/1,000 dilution.<br /><br />Lane 1: Hela cell lysate<br />Lane 2: PC-12 cell lysate<br />Lane 3: THP-1 cell lysate<br />Lane 4: K562 cell lysate<br /><br />Lysates/proteins at 10 µg/Lane.<br /><br />Predicted band size: 71 kDa<br />Observed band size: 100 kDa<br /><br />Exposure time: 1 minute;<br /><br />8% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1609-49" style="font-weight: bold;text-decoration: underline;">ET1609-49</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1:300,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of Phospho-FOXO3a (S253) on different lysates with Rabbit anti-Phospho-FOXO3a (S253) antibody (ET1609-49) at 1/1,000 dilution.

    Lane 1: Hela cell lysate
    Lane 2: PC-12 cell lysate
    Lane 3: THP-1 cell lysate
    Lane 4: K562 cell lysate

    Lysates/proteins at 10 µg/Lane.

    Predicted band size: 71 kDa
    Observed band size: 100 kDa

    Exposure time: 1 minute;

    8% 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 (ET1609-49) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-FOXO3a (S253) on different lysates with Rabbit anti-Phospho-FOXO3a (S253) antibody (<a href="/products/ET1609-49" style="font-weight: bold;text-decoration: underline;">ET1609-49</a>) at 1/1,000 dilution.<br /><br />Lane 1: 293T whole cell lysate<br />Lane 2: 293T treated with 50 uM LY294002 and 1 uM Wortmannin for 16 hours whole cell lysate<br /><br />Lysates/proteins at 15 µg/Lane.<br /><br />Predicted band size: 71 kDa<br />Observed band size: 100 kDa<br /><br />Exposure time: 20 seconds;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1609-49" style="font-weight: bold;text-decoration: underline;">ET1609-49</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1:200,000 dilution was used for 1 hour at room temperature.

    ☑ Cell treatment (CT)

    Western blot analysis of Phospho-FOXO3a (S253) on different lysates with Rabbit anti-Phospho-FOXO3a (S253) antibody (ET1609-49) at 1/1,000 dilution.

    Lane 1: 293T whole cell lysate
    Lane 2: 293T treated with 50 uM LY294002 and 1 uM Wortmannin for 16 hours whole cell lysate

    Lysates/proteins at 15 µg/Lane.

    Predicted band size: 71 kDa
    Observed band size: 100 kDa

    Exposure time: 20 seconds;

    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 (ET1609-49) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.

  • 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 ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1609-49" style="font-weight: bold;text-decoration: underline;">ET1609-49</a>, 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 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 ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1609-49" style="font-weight: bold;text-decoration: underline;">ET1609-49</a>, 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 ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1609-49" style="font-weight: bold;text-decoration: underline;">ET1609-49</a>, 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 ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1609-49" style="font-weight: bold;text-decoration: underline;">ET1609-49</a>, 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.

  • Immunocytochemistry analysis of NIH/3T3 cells labeling Phospho-FOXO3a (S253) with Rabbit anti-Phospho-FOXO3a (S253) antibody (<a href="/products/ET1609-49" style="font-weight: bold;text-decoration: underline;">ET1609-49</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-FOXO3a (S253) antibody (<a href="/products/ET1609-49" style="font-weight: bold;text-decoration: underline;">ET1609-49</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) 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.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of NIH/3T3 cells labeling Phospho-FOXO3a (S253) with Rabbit anti-Phospho-FOXO3a (S253) antibody (ET1609-49) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-FOXO3a (S253) antibody (ET1609-49) 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.

  • Flow cytometric analysis of NIH/3T3 cells labeling Phospho-FOXO3a (S253).<br /><br />Cells were fixed and permeabilized. Then stained with the primary antibody (<a href="/products/ET1609-49" style="font-weight: bold;text-decoration: underline;">ET1609-49</a>, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor&trade; 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (<a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

    Flow cytometric analysis of NIH/3T3 cells labeling Phospho-FOXO3a (S253).

    Cells were fixed and permeabilized. Then stained with the primary antibody (ET1609-49, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

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Citation

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