Thermo Scientific™ LanthaScreen™ PDCD4 HEK 293E Cell Line

Description

• The PI3K/AKT/mTOR pathway mediates signals for cell growth and survival, transcription and translation, and regulated cell death
• The significance of mTOR has been underscored recently by the identification of two distinct cellular complexes: mTORC1 (includes raptor and is rapamycin sensitive) and mTORC2 (includes rictor and is insensitive to rapamycin)
• Activation of the pathway by insulin (or other mitogens) leads to the phosphorylation of programmed cell death protein 4 (PDCD4) via mTORC1 (indirectly through the ribosomal protein p70 S6 kinase)
• LanthaScreen™ PDCD4 HEK293E is a human cell line which constitutively expresses GFP-PDCD4 fusion proteins
• This kinase target was introduced using lipid transfection followed by selection with Blasticidin
• These cells are a clonal population isolated by FACS using GFP fluorescence as a sorting marker
• A homogenous immunoassay ('addition only', no wash steps) was developed in 384-well format using a TR FRET-based detection method to monitor the phosphorylation of Ser457 on GFP-PDCD4 using a terbium-labeled phosphospecific antibody
• The assay has been optimized for performance under numerous experimental conditions (e.g., cell plating density, DMSO tolerance, insulin stimulation time, etc.) and displays excellent statistical data (Z' > 0.6)
• Moreover, this cell line has been validated with different stimuli and known inhibitors (shows correct EC50 and IC50 values) and serves as robust cellular readout for mTORC1 signaling.