Cellomics HCS Reagent Kit for detection of active forkhead box class O transcription factor FOXO1A.

FOXO1A primarily in cytosol	treat cells	FOXO1A translocated to nucleus	analyze	conclude

Description

The Cellomics FOXO1A Kit measures activation of FOXO1A, a transcription factor involved in the initiation of cell arrest and apoptosis. The kit contains a polyclonal rabbit FOXO1A antibody, a goat anti-rabbit secondary antibody conjugated to DyLight 549 Fluorophore and various other reagents and buffers required for immunofluorescent detection of FOXO1A for high-content screening (HCS) assays.

FOXO1A is a member of the forkhead box, class O transcription factors regulated by protein kinase B (AKT). In the absence of growth or survival signals, FOXO1A is active and resides in the nucleus, regulating transcription of FasL, p27kip, Bim, Bcl-6 and other genes critical for determining cell fate. Upon addition of growth factors or survival signals, FOXO1A is phosphorylated by AKT, which then primes its binding to the 14-3-3 protein. The inactive complex is then transported from the nucleus to the cytoplasm. The FOXO transcription factors promote cell survival, arrest and cell death, depending on the cell type and stimulus, and are potential targets for cancer therapeutics.

FOXO1A was activated using serum-starvation or with PI-3 kinase inhibitors in 3T3 L1 pre-adipocytes resulting in FOXO1A translocation from the cytoplasm to the nucleus (Figure 1). The FOXO1A assay was optimized with the Thermo Scientific ArrayScan® HCS Reader using the Compartmental Analysis BioApplication Software Module. FOXO1A can be quantitated using the nuclear intensity or the difference in intensity between the cytoplasm and the nucleus. Cells stained using this kit also can be imaged using fluorescence or confocal microscopy.

Figure 1. Serum starvation activates FOXO1A in 3T3 L1 pre-adipocytes. Cells were serum-starved for 24 hours and fixed and stained according to the kit protocol. Cells were labeled with Hoechst 33342 dye for nuclear staining and imaged using the ArrayScan VTI HCS Reader.

Figure 2. Dose response curves of FOXO1A in 3T3 L1 cells treated with LY294002 and wortmannin. Each data point represents eight wells. Curves in left panel represent separate data from three replicate plates. Single curve in right panel represents data from a single plate.

Related Poster Presentations

Kay Opperman, Krishna Vattem, and Richik N. Ghosh (2008) High-Content Analysis of Multiple Key Proteins in the AKT Signaling Pathway.

Kit Contents

Ordering Information

Screening size kits and components (bulk and custom) available with special pricing. Please call.

To order Cellomics HCS Reagent Kits, please call 800-874-3723 in the U.S.
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