Purification of XIAP-Binding Proteins

S10 cytoplasmic lysate from HeLa cells was prepared as described.¹⁷ (link) Briefly, HeLa-S cells (2 ml packed cell volume (PCV); Biovest International, Tampa, FL, USA) were resuspended in 2 ml of hypotonic buffer [10 mM Tris-HCl (pH 7.6), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT] containing EDTA-free protease inhibitor cocktail (Roche) and lysed using dounce homogenizer (30 strokes, pestle B). A mixture of 4 ml of HeLa S10 cytoplasmic lysate and 12 ml binding buffer [20 mM Tris (pH 7.6), 10 mM MgCl₂, 120 mM KCl, 8% sucrose, 2 mM DTT] containing EDTA-free protease inhibitor cocktail (Roche) and ribonuclease inhibitor (Promega) was incubated at 37°C for 10 min. An in vitro transcribed, strepto-tagged XIAP IRES RNA or strepto-tagged GAPDH RNA was added to the mixture and further incubated for 10 min at 37°C. RNA-dihydrostreptomycin affinity chromatography was performed as described^15,18 (link) and the RNA associated proteins were analyzed using protein gel blot analysis.