Leishmania donovani promastigotes strain AG83 (MHOM/IN/83/AG83) was grown in M199 medium (Sigma-Aldrich) supplemented with 10% FBS (Invitrogen) and penicillin–streptomycin mixture at 22 °C with slow shaking. To prepare GST-LdCyc1-CRK3 kinase complex, bacterially expressed GST-LdCyc1 was first bound to glutathione–sepharose beads (GE Healthcare Lifesciences), and the bead-bound GST-LdCyc1 was then incubated with an extract of Sf9 cells expressing LdCRK3 in the binding buffer (50 mM Tris-HCl, pH 8.0 containing

50 mM NaCl, 5 mM NaF, 1 mM Na3VO4, 0.1 mM EDTA, 0.1% Triton X-100, 10% LY294002 glycerol, 2 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl flouride (PMSF) and protease inhibitors) on a rotating wheel at 4 °C for overnight. The beads were washed three times with the same binding buffer, and the kinase complex was eluted with 50 mM Tris-HCl, pH 8.0, containing 10% glycerol, 10 mM reduced glutathione and PMSF. The complete ORF of LdHAT1 was cloned into pET21b vector, and the C-terminal 6His-tagged chimera was expressed in pG-JKE8 (TAKARA)-transformed Escherichia coli strain BL21 cells (expressing GroEL and GroES chaperons for greater solubility of the over-expressed protein) by induction

with 1 mM isopropyl β-D-1-thiogalactopyranoside at 37 °C Selleck Staurosporine for 3 h. Two mutants of LdHAT1, viz., LdHAT1ΔCy and LdHAT1-T394A were also cloned into pET21b vector and expressed as mentioned above. All 6His-tagged proteins were purified over Ni-NTA agarose beads. To perform interaction assay between LdCyc1 and LdHAT1, 5 μg of bacterially purified LdHAT1 protein was incubated on a rotating wheel at 4 °C for 1 h with glutathione beads bound to 0.2 μg of either GST or GST-LdCyc1 proteins in 50 mM Na-phosphate (pH 8.0) containing 250 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM EDTA, 2 mM DTT and protease inhibitors. Subsequently, the beads were washed six times with the same buffer, and the bound proteins were analysed by immunoblot analysis with appropriate antibodies. Similar experiments were carried out with the mutant LdHAT1 proteins. To synchronize L. donovani

promastigotes, exponentially growing cells were blocked with 10 mM hydroxyurea (HU) for 36 h followed by releasing until the arrest by re-suspending the cells in equal volume of growth medium, and cells were collected at different intervals. The synchronicity of cell cycle progression was confirmed by analysis in a flow cytometer (Supporting Information, Fig. S3). The population of cells from each time point was also examined by analysing the fluorescence and differential interference contrast (DIC) images of 4′,6-diamidino-2-phenylindole (DAPI)-stained cells captured by a Zeiss Axio-observer Z1 inverted microscope (Fig. S3). Cells from different time intervals were lysed in 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 0.