FKHR (for forkhead in rhabdomyosarcoma), FKHRL1, and AFX1 are members of a subfamily of the forkhead family of transcription factors. AFX1, also known as FoxO4, is expressed in a wide variety of tissues and, like other FKHR proteins, AFX1 contains a single forkhead domain and serine-proline-rich region, which mediate DNA binding. AFX1-mediated transcriptional activation is regulated by the serine/threonine kinase Akt1, which phosphorylates AFX1 and in turn, sequesters AFX1 in the cytosol, thereby blocking nuclear localization and DNA binding. Genetic mutations in FKHR genes, including the t(2;13) and t(1;3) translocations, are commonly found in alveolar rhabdomyosarcomas. Additionally, the t(x;11) translocation of the AFX1 gene, which involves the fusion of a serine-proline-rich sequence of AFX1 to the carboxy terminus of a truncated MLL, results in acute lymphocytic leukemia.
Background References
1. Xiong, S. et al. 2011. FoxO1 mediates an autofeedback loop regulating SIRT1 expression. J. Biol. Chem.. 286: 5289-5299.
2. Noh, TW. et al. 2009. Predicting recurrence of nonfunctioning pituitary adenomas. J. Clin. Endocrinol. Metab.. 94: 4406-4413.
Tissue Specificity
Heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. Isoform zeta is most abundant in the liver, kidney, and pancreas.
Post-translational Modification
Acetylation by CREBBP/CBP, which is induced by peroxidase stress, inhibits transcriptional activity. Deacetylation by SIRT1 is NAD-dependent and stimulates transcriptional activity.; Phosphorylation by PKB/AKT1 inhibits transcriptional activity and is responsible for cytoplasmic localization. May be phosphorylated at multiple sites by NLK.; Monoubiquitinated; monoubiquitination is induced by oxidative stress and reduced by deacetylase inhibitors; results in its relocalization to the nucleus and its increased transcriptional activity. Deubiquitinated by USP7; deubiquitination is induced by oxidative stress; enhances its interaction with USP7 and consequently, deubiquitination; increases its translocation to the cytoplasm and inhibits its transcriptional activity. Hydrogene-peroxide-induced ubiquitination and USP7-mediated deubiquitination have no major effect on its protein stability.
Western blot analysis of FOXO4 on different lysates with Rabbit anti-FOXO4 antibody (ET1612-66) at 1/1,000 dilution.
Lane 1: HeLa cell lysate Lane 2: PC-12 cell lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 54 kDa Observed band size: 54 kDa
Exposure time: 25 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 (ET1612-66) at 1/1,000 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.
Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue using anti-FOXO4 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 (ET1612-66, 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-FOXO4 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 (ET1612-66, 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-FOXO4 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 (ET1612-66, 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 placenta tissue using anti-FOXO4 antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (ET1612-66, 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.
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