, After the incubation period, cells (1 × 10 7 cells) were sedimented, washed twice with PBS and suspended in 1 ml of PBS. Aliquots of the suspension (2 × 10 5 cells) were applied on microscopic slides and air-dried. After fixing the cells for 2 min in ice-cold methanol, the slides were air-dried for 20 min. Non-adherent cells were removed by gentle washing (0.1% Triton X-100 in PBS) followed by, Fluorescence microscopy. Promastigote cells were incubated for 72 h without RAD or with the IC 90 of RAD

, :25). After washing the slides thrice, Mowiol and coverslips were applied and the slides were left to dry for 24 h at 4 °C. Fluorescence microscopy was carried out on an EVOS FL Auto epifluorescence microscope, Triton-X 100 in PBS). Slides were then incubated for 1 h with monoclonal mouse anti-tubulin

;. Phosphoproteomics-;-pen/strep, . Gibco, and . Uk), Cultures were incubated at 34 °C/5% CO 2 for 72 h. Parasites were harvested by centrifugation at 2000 × g, 4 °C, and washed in 1) ice-cold wash buffer (21 mM HEPES pH 7.5, 137 mM NaCl, 5 mM KCl) and 2) ice-cold wash buffer with protease and phosphatase inhibitors (1 mM Na-orthovanadate, 0.1 µM okadaic acid, 10 mM NaF, 10 mM o-phenanthroline, EDTA-free protease inhibitors). For lysis, parasites were resuspended in a solution of 7 M urea, 2 M thiourea, 40 mM Tris, 1% n-octyl-?-D-glycopyranoside, 1 mM MgCl 2 , 1 mM o-phenanthroline, 300 U benzonase, 1 mM Na-pervanadate (Na-orthovanadate activated in 18% H 2 O 2 ), protease inhibitors (Roche EDTA-free protease inhibitor tablets), and phosphatase inhibitor cocktails (P2850 and P5726 from Sigma), and sonicated for 3 × 15 s on ice. Lysates were incubated at ?80 °C for 30 min prior to reduction (50 mM DTT) and alkylation with 50 mM iodoacetamide. Proteins were precipitated in an 8-fold excess of ice-cold acetone-ethanol, 1:1, v/v, by overnight incubation at ?20 °C. Protein precipitates were reconstituted in 6 M urea/2 M thiourea and diluted in 50 mM NH 4 HCO 3 for digestion with trypsin at a 1:75 enzyme-substrate ratio overnight. Digestion was quenched by addition of formic acid (FA) to a final concentration of 1%. Aliquots of 330 µg digested proteins were used for phosphopeptide enrichment by TiO 2 (Titansphere, 5 µm, Leishmania mexicana MNYC/BZ/62/M379 promastigotes were cultured in SDM-79 medium 93 supplemented with 10% heat-inactivated FCS

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