mTOR Translocation Visualization in TSC2-/- p53-/- MEFs

Immunofluorescence to detect mTOR translocation was performed as previously described [23 (link)]. TSC2^−/−p53^−/− MEFs (kindly provided by Dr. John Blenis, Weill Cornell Medical College) were seeded onto fibronectin-coated chamber slides. Cells were starved for serum over night and deprived of amino acids for 4 h using a media without any amino acids. After re-stimulating with amino acid for 1 h, the cells were fixed with 4% formaldehyde, and, subsequently, permeabilized using 0.05% saponin in PBS. Slides were treated with blocking solution (5% bovine serum albumin), and incubated with primary antibodies (anti-mTOR: Cell signaling, anti-LAMP1: BD pharmingen, San Jose, CA, USA) overnight at 4 °C followed by secondary antibodies conjugated with Alexa488 and Alexa568. Images were captured with Carl Zeiss LSM700 confocal laser scanning microscope and measured using ZEN microscope software.