Fc203b

iTRAQ labelled peptides were then fractionated by cation exchange chromatography using high performance liquid chromatography (Waters, Inc, Milford, MA) with a Zorbax 300 SCX column (5 μm; 2.1 mm × 150 mm, Agilent, USA). Lyophilized peptides were reconstituted in 2 ml (1 ml for 4-plex) loading buffer (buffer A consisting of 10 mM KH₂PO₄ in 75:25 water: acetonitrile, pH 2.9). The flow rate was kept at 0.4 ml/min for 17 minutes for loading after which it was increased to 0.8 ml/min. The following gradient program was employed: 0% Buffer B (10 mM KH₂PO₄, 1 M KCl in 75:25 water: acetonitrile (pH 2.7) for 17 min, 0% to 50% Buffer B in 39 min, 50% to 100% Buffer B in 5 min, 100% Buffer B for 10 min, 100% to 0% Buffer B in 8 min, and 0% Buffer B for 10 min (Supplementary Fig. 1). Eluting peptides were monitored at 214 nm and 20 fractions were collected using a fraction collector (FC 203B, Gilson). These fractions were then lyophilized by centrifugal evaporation (eppendorf).

The fraction of ribosome was separated by centrifugation in a sucrose gradient. Cells pretreated with 100 μg/ml cycloheximide were lysed in 1 ml lysis buffer (10 mM Tris, pH 7.4, 150 mM KCl, 5 mM MgCl2, 100 μg ml^–1 CHX, 0.5% Triton-X-100, freshly add 1:100 protease inhibitor, 40 U ml^–1 SUPERasin). After centrifugation at 15,000 g for 15 min, the supernatant was separated by 5/50% w/v sucrose gradient at 4 °C for 4 h at 140, 000 g (Beckman, rotor SW28). The sample was then fractioned and analyzed by Gradient Station (BioCamp) equipped with ECONO UV monitor (BioRad) and fraction collector (FC203B, Gilson). The fractions resulting from sucrose gradient were used for RNA extraction and qRT-PCR.

Cells were incubated with 100 μg/ml cycloheximide (CHX) per 10 cm dish at 37 °C for 10 min. Then, the cells were collected and lysed on ice with lysis buffer for 3 min. After centrifugation at 12000 × g for 5 min at 4 °C, the supernatant was loaded onto a 15/50% (w/v) sucrose gradient solution prepared in lysis buffer. Centrifugation was performed at 4 °C for 2 h 40 min at 27,400 × g (Beckman, SW40 rotor). The RNA content was then fractioned and measured using a Gradient Station (BioCamp) coupled with a UV monitor (Bio-Rad) and a fraction collector (FC203B, Gilson). After fractionation, RNA was extracted using TRIzol and analyzed by qRT-PCR.

The striatal slices of the rat were incubated in the presence of 10 µCi of [³H]dopamine added to 1.5 mL oxygenated (95% O₂/5% CO₂, pH 7.4) and preheated (37 °C) Krebs bicarbonate buffer for 30 min [47 (link)]. The superfused striatal tissues were transferred into tissue chambers (in a volume of 0.3 mL), after loading with [³H]dopamine, and superfused with aerated and preheated Krebs bicarbonate buffer (Experimetria Kft, Budapest, Hungary). The flow rate was maintained at 1 mL/min by using a Gilson peristaltic pump (type M312, Villiers-Le Bel, France). The superfusate was discarded during the first 60 min preperfusion period, then, twenty-five fractions (3 min each) were collected by a fraction collector (Gilson type FC-203B, Middletown, WI, USA). Electrical field stimuli (parameters: 40 V voltage, 10 Hz frequency, 2 msec impulse duration for 3 min in fractions 4 and 18) were delivered by a Grass S88 Electrostimulator (Quincy, MA, USA) to induce vesicular [³H]dopamine release. The drugs were added to the striatal slices between the first and second electrical stimulations, as was indicated in the figure legends.

Prostate cancer cells were treated with cycloheximide (CHX) at 100 μg/ml for 2 minutes before collection. Cells were lysed on ice, and then centrifuged. Next, collect the supernatant and load onto a 10/50% w/v sucrose gradient. The gradients were centrifuged at 4°C for 4 hours at 27,500 rpm (Beckman, rotor SW28). Subsequently, the samples were fractioned and analyzed by Gradient Station (BioCamp) equipped with an ECONO UV monitor (BioRad) together with a fraction collector (FC203B, Gilson). The fractions were isolated total RNA by TRIzol reagent for RT-qPCR analysis.