TULP3 Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of TULP3
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

The Tubby protein family includes five members, namely the human homolog of murine Tubby, named TUB, and the Tubby-like proteins (TULPs). All Tubby proteins are expressed in the retina, but TUB and TULP3 are also widely distributed in the central nervous system (CNS). TUB is implicated in obesity whereas TULP-3 is required for neural development. Tubby proteins are suggested to function in transcription and signaling stimulated by G-protein activation. Tubby proteins bind phosphatidylinositol and localize to the plasma membrane. Activation of G protein-coupled receptors (GPCRs) and subsequent signaling via the Gq subclass of Gα proteins releases Tubby proteins from the plasma membrane by phospholipase C-mediated hydrolysis of PtdIns(4,5)P2. Following, the release from the membrane, Tubby proteins translocate to the nucleus.

Figure 1. Nuclear translocation of EGFP-TULP3. Cells were untreated (DMSO control, left panel), treated with 50 nM PMA (center panel), or treated with 100 nM isoproterenol for 2 hr (right panel). Arrows indicate the nuclear localization of EGFP-TULP3 that is detected by the image analysis algorithm.

Figure 2. Concentration response curves in the TULP3 assay: A) PMA concentration response. The EC₅₀ is approximately 2 nM. Concentration response was measured in 9 point half log dilution series (n=16). Cells were treated with PMA for 2 hr. Cells were then fixed and nuclear translocation was measured using the Cellomics ArrayScan V^TI Reader and the Redistribution V3 BioApplication. % activity was calculated relative to the positive (50 nM PMA) and negative control (0.25% DMSO). B) Isoproterenol concentration response. The EC₅₀ is approximately 3 nM. Concentration response was measured in 9 point half log dilution series (n=4). Cells were analyzed using the IN Cell Analyzer 3000 (GE Healthcare).