Gradient master former

The cells were treated for 10 min with 100 μg/ml cycloheximide (Sigma) 48 h post-transfection. After washing with PBS containing cycloheximide, they were mechanically disrupted with a Dounce homogenizer in buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT) containing cycloheximide. The cytoplasmic fraction was recovered by centrifugation for 10 min at 1000 g and at 4°C, and then further clarified by two successive centrifugations at 10 000 g. A volume corresponding to 1 mg total proteins was loaded on 10–50% (w/w) sucrose gradients, prepared with a Gradient Master former (BioComp Instruments). After centrifugation at 4°C and at 36 000 rpm for 105 min in a SW41 rotor (Optima L100XP ultracentrifuge; Beckman Coulter), the fractions were collected at OD_{254 nm} with a Foxy Jr. gradient collector (Teledyne Isco).

Cells were incubated for 10 min in Dulbecco's-modified Eagle's medium (DMEM) containing 100 μg/ml cycloheximide (CHX) (Sigma-Aldrich). All subsequent steps were performed in the presence of 100 μg/ml CHX. Cells were collected by centrifugation at 4°C (5 min, 300 × g) after treatment with trypsin and CHX. Cell pellets were mechanically disrupted with a Dounce homogenizer in 800 μl of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT) containing CHX. The lysate was cleared from nuclei by centrifugation at 4°C (10 min, 1000 × g), and the upper cytoplasmic fraction was further clarified by two successive centrifugations at 10 000 × g, 5 min each. An aliquot containing 1 mg protein was loaded on a 10–50% (w/w) sucrose gradient prepared in buffer A, which was generated with a Gradient Master former (BioComp Instruments). The gradient was centrifuged at 4°C (2 h 15 min, 36 000 rpm), using a SW41Ti rotor (Optima L100XP ultracentrifuge; Beckman-Coulter), and the fractions were collected with a Density Gradient Fractionation System (Teledyne Isco) with continuous reading of OD_254nm.