FKHR (for forkhead in rhabdomyosarcoma) and FKHRL1 are members of the forkhead family of transcription factors. Transcriptional activation of FKHR proteins is regulated by the serine/threonine kinase Akt1, which phosphorylates FKHRL1 and results in FKHRL1 associating with 14-3-3 proteins and being retained in the cytoplasm. Induction of apoptosis or withdrawal of growth factors stimulates dephosphorylation and nuclear translocation of FKHR proteins, leading to FKHR-induced gene-specific transcriptional activation. FKHR, also designated forkhead box protein O1A (FOXO1), is a ubiquitously expressed protein that shuttles between the cytoplasm and nucleus. Genetic mutations in FKHR genes, including the t(2;13) and t(1;3) translocations, are commonly found in alveolar rhabdomyosarcomas. These translocations result in the fusion of the amino terminus of Pax-3 or Pax-7, including the paired box and homeodomain DNA-binding domains, with the carboxy-terminus of FKHR, which contains a transcriptional activation domain. The Pax-3/FKHR fusion protein appears to function as an oncogenic transcription factor that enhances the activation of normal Pax-3 target genes.
Background References
1. Xiao N et al. The E3 ubiquitin ligase Itch is required for the differentiation of follicular helper T cells. Nat Immunol 15:657-66 (2014).
2. Chen C et al. High cytoplasmic FOXO1 and pFOXO1 expression in astrocytomas are associated with worse surgical outcome. PLoS One 8:e69260 (2013).
Immunocytochemistry analysis of Hela cells labeling FOXO1A with Rabbit anti-FOXO1A antibody (HA720161F) at 1/50 dilution.
Cells were fixed in 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes, and then blocked with 2% normal goat serum for 1 hour at 37 ℃. Cells were then incubated with Rabbit anti-FOXO1A antibody (HA720161F) at 1/50 dilution in 2% normal goat serum overnight at 4 ℃. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/200 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) were used as the secondary antibody at 1/800 dilution.
Flow cytometric analysis of NIH/3T3 cells labeling FOXO1A.
Cells were fixed and permeabilized. Then incubated for 30 minutes at +4℃ with FOXO1A (HA720161F, red, 1ug/ml) and Rabbit IgG Isotype Control (iFluor™ 488, green, 1ug/ml). Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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