LanthaScreen™ PRAS40 293E Cell Line

Description

• Gene Symbol: PRAS40
• Primary Agonist: Insulin
• Antagonists: IGF-1
• The PI3K/AKT pathway is activated by various stimuli (including insulin and IGF-1) and mediates signals for cell growth, cell survival, translation, and inhibition of apoptosis.
• PRAS40 (Proline-rich AKT substrate, 40 kDa) is a cytosolic protein that is phosphorylated at position Thr246 by AKT directly upon stimulation of the pathway.
• PRAS40 has been shown to bind to the mTORC1 complex and inhibit downstream signaling (i.e., phosphorylation of 4E-BP1 or p70S6K).
• LanthaScreen™ PRAS40 HEK293E is a human cell line which constitutively expresses GFP-PRAS40 fusion proteins.
• The kinase substrate was introduced using lentiviral transduction followed by selection with blasticidin.
• This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as a sorting marker.
• The phosphorylation of PRAS40 at Thr246 is detected in cell lysates using a terbium-labeled phosphospecific antibody in a time-resolved FRET (TR-FRET) readout.
• The performance of this assay has been tested using numerous experimental variables, including cell plating density, stimulation time, DMSO tolerance, and antibody incubation time.
• Under optimized conditions, the assay has been further validated and shows correct EC₅₀ and IC₅₀ values, with insulin as the primary agonist and PI-103 as the known inhibitor.
• The assay displays excellent statistical data (Z'-factor >0.6), high signal-to-background (response ratio), and is a robust cell-based readout of AKT signaling.