Jurkat cells (1 × 107) were collected and washed three times with PBS before lysis by sonication in 1 ml of fractionation buffer [250 mM sucrose, 20 mM Hepes (pH 7.4), 10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA, protease cocktail inhibitor (Santa Cruz Biotechnology), 10 mM NaF, and 1 mM Na3OV4]. Samples were then centrifuged at 10,000g for 10 min, and the supernatant was transferred to a fresh ultracentrifuge tube. The sample was spun in a Beckman L8-80M centrifuge at 100,000g for 1 hour at 4°C. The subsequent pellet was washed once in 500 μl of fractionation buffer before being resuspended in sterile PBS.

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