L5750

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).

Cells were washed twice with ice cold PBS and lysed with 2 x sample buffer (125 mM Tris-Cl, pH 6.8 [Sigma, T1378], 4% SDS [Sigma, L-5750], 20% glycerol [Sigma, G5516], 200 mM DTT [Merck, D0632], 0.004% bromophenol blue) supplemented with protease inhibitor cocktail (cOmplete, EDTA free; Roche, 05056489001) and for signaling experiments supplemented with phosphatase inhibitors (phosSTOP; Roche, 04906837001). Lysates were subjected to SDS-gel electrophoresis on 10% or 4-20% gradient gels (mini- or midi-PROTEAN TGX; Bio-Rad, 5671095, 5671035, 5671034, 5671094, 4561036, 4561095). Proteins were transferred to PVDF membranes (TransBlot® Turbo^TM LF PVDF; Bio-Rad, 170-4275, 170-4274) followed by blocking and antibody incubation in 2% BSA (Merck, BSAV-RO 10735094001) in Tris-buffered saline (pH 7.5), with 0.05% Tween-20 (Sigma, P1379). Membranes incubated with fluorescent secondary antibodies (LI-COR) were developed using an Odyssey infrared scanner (LI-COR), or incubated with horseradish peroxidase-conjugated antibodies and developed using a Clarity Western ECL substrate solution (Bio-Rad, 1705060) and ChemiDoc XRS+ imaging system (Bio-Rad). Quantification and analysis of immunoblots was performed with Odyssey Software (version 3.0.30) or ImageJ (version 1.52p).