Silencing Raptor, Rictor, DEPTOR and Nedd4-2 in PHT Cells

Dharmafect 2 transfection reagent (Thermo Scientific, Rockford, IL) and small interfering RNAs (siRNAs) (Sigma-Aldrich, St. Louis, MO), targeting raptor (100 nM; sense, 5′CAGUUCACCGCCAUCUACA), rictor (100 nM; 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 (100 nM; 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 h31 (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.