Silencing mTOR Pathway Regulators

Dharmafect 2 transfection reagent (Thermo Scientific, Rockford, IL) and small interference RNAs (siRNAs) (Sigma-Aldrich, St. Louis, MO), targeting raptor (100 nM; sense, 5′CAGUUCACCGCCAUCUACA), rictor (100nM; sense, 5′ CGAUCAUGGGCAGGUAUUA), DEPTOR (SASI_1297010-H/5582, 1297011-H) or Nedd4-2 (100 nM; 5′ CCCUAUACAUUUAAGGACU) were used. Control cells were transfected with a non-coding scrambled sequence (100nM; sense: 5′GAUCAUACGUGCGAUCAGATT). siRNAs were added to cultured PHT cells (~3.75 × 10⁶ cells/well in 6 well plate; ~ 7.5 × 10⁶ cells in 60 mm dish) after 18 h in culture, incubated for 24 h [39 (link)] and subsequently removed and replaced by fresh medium. At 90 hours in culture, efficiency of target silencing was determined at the protein and functional levels using Western blot.