Homogeneize buffer with halt protease and phosphatase inhibitor cocktail 1x

The Plasma Membrane Protein Extraction Kit (Abcam) was used according to manufacturer’s specifications. Briefly, tissues were washed in ice-cold phosphate buffered saline (PBS) 1X as many times were needed to eliminate most of the blood present. The tissue was homogenized in 2 mL of ice-cold Homogeneize Buffer with Halt Protease and Phosphatase Inhibitor Cocktail 1X (Thermo) in a BeadBug Microtube homogenizer (Benchmark). The homogenate was centrifuged at 700 g during 10 min at 4 °C to collect tissue and cells that were not lysed. The supernatant was transferred to a new tube and was centrifuged at 10,000 g during 30 min at 4 °C. Pellet formed correspond to all membrane proteins and supernatant correspond to cytosolic proteins. Cytosolic proteins were precipitated with four volumes of 95% acetone and centrifuged [16 ].

The Plasma Membrane Protein Extraction Kit (Abcam, Cambridge, UK) was used according to manufacturer’s specifications. Briefly, tissues were washed in ice-cold phosphate buffered saline (PBS) 1X as many times were needed to eliminate most of the blood present. The tissue was homogenized in 2 mL of ice-cold Homogeneize Buffer with Halt Protease and Phosphatase Inhibitor Cocktail 1X (Thermo, Waltham, MA, USA) in a BeadBug Microtube homogenizer (Benchmark, Sayreville, NJ, USA). The homogenate was centrifuged at 700× g for 10 min at 4 °C to collect tissue and cells that were not lysed. The supernatant was transferred to a new tube and was centrifuged at 10,000× g during 30 min at 4 °C. Pellet formed correspond to all membrane proteins and supernatant correspond to cytosolic proteins. Cytosolic proteins were precipitated with four volumes of 95% acetone and centrifuged.