Phospho-FoxO1 (T24)/FoxO3a (T32) Antibody [M8K9]

Application: Reactivity: Mouse, Human

Usage Information

Dilution
WB1:1000 IF1:1000 FCM1:1000

Application
WB, IF, FCM

Reactivity
Mouse, Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
69 kDa 55-150 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight.

Positive Control	NIH/3T3 cells (starved, 24 h; Calyculin A, 100nM, 30 min); Jurkat cells (Calyculin A, 100nM, 40 min)
Negative Control	Jurkat cells (LY294002, 20μM, 2 h); Jurkat cells; NIH/3T3 cells (starved, 24 h)

Datasheet & SDS

Biological Description

Specificity
Phospho-FoxO1 (T24)/FoxO3a (T32) Antibody [M8K9] detects endogenous levels of total FoxO1 and FoxO3a protein only when it is phosphorylated at T24 and T32, respectively.

Clone
M8K9

Synonym(s)
Forkhead box protein O1; Forkhead box protein O1A; Forkhead in rhabdomyosarcoma; FOXO1; FKHR; FOXO1A

Background
Phospho-FoxO1 (T24)/FoxO3a (T32) refers to the phosphorylated forms of FoxO1 at threonine 24 and FoxO3a at threonine 32, key sites within the Forkhead box O (FoxO) family of transcription factors that regulate cell proliferation, apoptosis, metabolism, stress resistance, and longevity. These proteins feature a conserved winged-helix DNA-binding domain, nuclear localization/export signals, and transactivation domains, with the N-terminal region containing 14-3-3 binding motifs where T24/T32 phosphorylation occurs. Phosphorylation by AKT at these residues (T24 in FoxO1, T32 in FoxO3a) promotes 14-3-3 protein binding, leading to nuclear exclusion, cytoplasmic retention, and inhibition of transcriptional activity, thereby suppressing tumor suppressor functions like cell cycle arrest via p21/p27 and apoptosis via Bim/TRAIL/PUMA. This inactivation occurs via the insulin/PI3K/AKT pathway, countering active nuclear FoxO's promotion of stress resistance genes (e.g., SOD, catalase) and autophagy under nutrient deprivation or oxidative stress. In disease contexts, such phosphorylation links to cancer progression , fibrosis, and metabolic disorders by disrupting FoxO-mediated homeostasis. Conversely, AMPK opposes this by phosphorylating nearby sites , hierarchically blocking T24 phosphorylation through steric/electrostatic hindrance and reducing 14-3-3 affinity, thus sustaining FoxO activity.

Background

Phospho-FoxO1 (T24)/FoxO3a (T32) refers to the phosphorylated forms of FoxO1 at threonine 24 and FoxO3a at threonine 32, key sites within the Forkhead box O (FoxO) family of transcription factors that regulate cell proliferation, apoptosis, metabolism, stress resistance, and longevity. These proteins feature a conserved winged-helix DNA-binding domain, nuclear localization/export signals, and transactivation domains, with the N-terminal region containing 14-3-3 binding motifs where T24/T32 phosphorylation occurs. Phosphorylation by AKT at these residues (T24 in FoxO1, T32 in FoxO3a) promotes 14-3-3 protein binding, leading to nuclear exclusion, cytoplasmic retention, and inhibition of transcriptional activity, thereby suppressing tumor suppressor functions like cell cycle arrest via p21/p27 and apoptosis via Bim/TRAIL/PUMA. This inactivation occurs via the insulin/PI3K/AKT pathway, countering active nuclear FoxO's promotion of stress resistance genes (e.g., SOD, catalase) and autophagy under nutrient deprivation or oxidative stress. In disease contexts, such phosphorylation links to cancer progression , fibrosis, and metabolic disorders by disrupting FoxO-mediated homeostasis. Conversely, AMPK opposes this by phosphorylating nearby sites , hierarchically blocking T24 phosphorylation through steric/electrostatic hindrance and reducing 14-3-3 affinity, thus sustaining FoxO activity.

References
https://pubmed.ncbi.nlm.nih.gov/31308176/ https://pubmed.ncbi.nlm.nih.gov/25225602/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%