Ua 6 uv vis

Polysome profiling of individual mRNAs was described before (8 (link)), with the exception that total lysates were used. Macrophages were rinsed in ice cold 1× PBS containing 0.1 mg/ml cycloheximide prior to lysis. Total lysates from 10 × 10⁶ cells were separated on a linear 10–50% sucrose gradient by ultracentrifugation for 2 h at 35 000 rpm using a SW40.1 Ti rotor (Beckman-Coulter). After centrifugation 12 fractions (1 ml each) per gradient were collected using a UA-6 UV/VIS (Teledyne/ISCO Inc.) device. The RNA of each fraction was precipitated overnight with isopropanol and 3 M Na-acetate at –20°C and purified using the NucleoSpin RNA kit (Macherey+Nagel) by dissolving the resulting pellet in buffer RA1 from the kit. Purified RNAs were subjected to cDNA synthesis and qRT-PCR as described above.

For polysome profile analysis, 500 mL of each PTP-TAP T. brucei culture were grown overnight at 28°C to mid-log phase and cells were treated with cyclohexamide 100 μg/mL for 5 minutes. The cultures were immediately chilled on ice and collected by centrifugation at 3,000 X g at 4°C for 7 minutes. Cells were washed twice with ice-cold Salts buffer (Tris-HCl 10 mM pH 7,5; KCl 30 mM; MgCl₂; 10 mM; DTT 1 mM; cyclohexamide 100 μg/mL), pellet volume was estimated and suspended with the same volume of Lysis buffer (Tris-HCl 10 mM pH 7,5; KCl 30 mM; MgCl₂; 10 mM; DTT 1 mM; cyclohexamide 100 μg/mL; 1,2% Triton), supplemented with 1X Protease cOmplete inhibitor cocktail (Roche). Lysates were clarified by centrifugation at 17,000 X g for 15 minutes. Eight hundred micrograms equivalent of OD₂₆₀ units was loaded on a 7–47% sucrose gradient prepared in Salts buffer and centrifuged at 39,000 RPM for 2 hours at 4°C in a Beckman SW41Ti rotor. The gradients were fractionated by upward displacement with 60% (w/v) sucrose using a gradient fractionator ISCO UA-6 UV Vis with Type 11 optical unit at 254 nm and fractions were collected manually for subsequent western blotting analysis. Subunit profile analysis was performed as described before with modifications; lysis buffer was supplemented with 50 mM of EDTA and lysed was centrifuged on a 5–25% sucrose gradient for 4 hours.