Leishmania extracts

Cytoplasmic fraction 24205340 /13

Cultures containing 1.4x10⁷ promastigotes/ml were pelleted were lysed in ice-cold buffer A (20 mM Tris–HCl pH 7.6, 1 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM benzamidine, 2 mM sodium molybdate, 2 mM sodium β-glycerophosphate, 0.2 mM sodium orthovanadate, 120 mM KCl, 1 μg/ml leupeptin and pepstatin A, and 10 μg/ml antipain) containing 1% Triton X-100 and then centrifuged at 17000 x g for 20 min in order to obtain a cytoplasmic fraction.

Fraction enriched in nuclear proteins

The pellet was resuspended in RIPA buffer (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin) and centrifugated at 17000 x g for 20 min, to obtain a fraction enriched in nuclear proteins.