A High-Throughput, Cell-Based Screening Method for siRNA and Small Molecule Inhibitors of mTORC1 Signaling Using the In Cell Western Technique

• DOI: 10.1089/adt.2009.0213
• Publication Date: 2010-01-19T11:10:07Z
• Authors: Gregory R. Hoffman; Nathan J. Moerke; Max Hsia; Caroline E. Shamu; John Blenis
• Keywords: Cell Signaling; Lymphangioleiomyomatosis

The mTORC1 pathway is a central regulator of cell growth, and defective regulation plays causative role in variety human diseases, including cancer, tumor syndromes such as the tuberous sclerosis complex (TSC) lymphangioleiomyomatosis (LAM), metabolic diseases diabetes obesity. Given importance signaling these there has been significant interest developing screening methods suitable for identifying inhibitors activation. To this end, we have developed high-throughput, cell-based assay detection rpS6-phosphorylation measure signaling. This takes advantage "In Cell Western" (ICW) technique using Aerius infrared imaging system (LI-COR Biosciences). ICW procedure involves fixation immunostaining cells manner similar to standard immunofluorescence but secondary antibodies conjugated infrared-excitable fluorophores quantitative by scanner. In addition, are stained with an succinimidyl ester dye, which covalently modifies free amine groups fixed provides number. We present validation data pilot screens 384-well format demonstrating that statistically robust method both small molecule siRNA approaches designed identify