Immunoblotting of Transfected HEK293 Cells

Immunoblot experiments were conducted by following the protocol described in Cáceres et al. (2015) (link). Briefly, transfected HEK293 cells were washed once with ice-cold DPBS and lysed with 150 μL of ice-cold lysis buffer containing 1% (v/v) Triton X-100 (Merck, 108603), 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 7.4), 1 mM sodium orthovanadate, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma, 78830), and protease inhibitor cocktail (Cytoskeleton, Inc., PIC02) for 10 min at 4°C. The lysates were centrifuged at 12,000 g at 4°C for 10 min. The supernatants were mixed with 150 μL of 2x Reducing Sample Buffer [RSB: 60 mM Tris-HCl pH 6.8, 25% (v/v) glycerol, 2% (w/v) SDS (Sigma, L5750), 14.4 mM 2-mercaptoethanol, 0.1% (w/v) bromophenol blue] and size-fractionated by 7.5% SDS-PAGE. Lauryl sulfate (Sigma, L5750) was the form of SDS used in all gel solutions. The immunoblots were visualized by Pierce ECL Western Blotting Substrate (ThermoFisher, 34080) and images were acquired with a MiniHD9 imager (Uvitec).