Nuclear fractionation and neomycin extraction

Nuclei were isolated according to a method by Mukai et al. [58] with some modifications. Cells were washed two times in PBS and once briefly in buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 340 mM sucrose, 10% glycerol). Cells were resuspended in buffer A containing 0.1% Triton X-100, 1 mM DTT, 5 μg/ml leupeptin and 5 μg/ml aprotinin, left to swell for 5 min on ice and centrifuged at 1300 g for 5 min at 4°C. Nuclei were washed quickly with retention buffer (20 mM Tris pH 7.5, 70 mM NaCl, 20 mM KCl, 5 mM MgCl₂ and 3 mM CaCl₂ [59]). Nuclei were incubated twice in retention buffer for 30 min at room temperature, split into two equal fractions and further incubated in the presence or absence of 5 mM neomycin (trisulfate salt, Sigma N6386) for 30 min at room temperature. Samples were centrifuged at 9600 g for 5 min at 4°C and super-natants were collected. For lipid overlay assays, neomycin supernatants were dialysed twice against 20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM EDTA and 0.1% NP-40.