Fractionation of trypanosome cellular extracts was performed as described previously [77]. The integrity of the cellular compartment was confirmed by using antibodies directed against the cytosolic protein Hsp70 or the nuclear RNA polymerase II [78]. Immunoprecipitation of TbLpn from T. brucei cytosolic extracts As it was previously determined that TbLpn is localized in the cytosol,

immunoprecipitation of TbLpn was performed using PF form T. brucei cytosolic extracts. Ten μg of purified anti-TbLpn antibodies or 10 μl of IP buffer (for mock immunoprecipitations) (20 mM Hepes [pH 7.9], 150 mM sucrose, 150 mM KCl, 3 mM MgCl2, 0.5% Nonidet- P40, 1 μg/ml of pestatin A, 1 μg/ml of leupeptin, 5 mM PMSF) were added to 200 μl of cytosolic extract in a final volume of 300 μl of IP buffer. The samples were incubated at 4°C for at least 12 h with

gentle rotation. Ten μl of Protein A-Sepharose (GE Healthcare) was then added, and the samples incubated 1 hour at 4°C with gentle RO4929097 supplier rotation. Immune complexes were recovered by centrifugation at 3,000 × g for 30 s and washed five times, each time for 5 min, with 1 ml of IP buffer. Phosphatidic acid phosphatase assays The standard reaction contained 50 mM Tris–HCl buffer (pH 7.5), 1 mM MgCl2, and 0.4 mM 1,2-dioctanoyl-sn-glycero-3-phosphate (DiC8 LEE011 PA) (Avanti Polar Lipids) in a total volume of 50 μl. Reactions were initiated by the addition of recombinant proteins (50–250 ng), and carried out in triplicate at 30°C for 30 min. The reaction was terminated by the addition of 100 μl of PiBlue reagent (BioAssay Systems), and the color allowed to Ergoloid develop at room temperature for 30 minute. The absorbance was measured with a spectrophotometer at 620 nm. The amount of phosphate produced was quantified from a standard curve using 0.5–4 nmol of potassium phosphate. The reactions were linear with time and protein concentration. The enzymatic activity was expressed as the number of pmol of phosphate released per minute. Acknowledgments We thank Dr. Laurie K. Read (University at Buffalo, Department of Microbiology and Immunology) for providing several reagents essential to the completion of many experiments. We are also

grateful to Dr. Adam Rich (The College at Brockport, Department of Biology) for helpful discussions. References 1. Bachand F: Protein arginine methyltransferases: from unicellular eukaryotes to humans. Eukaryot Cell 2007, 6:889–898.PubMedCrossRef 2. Bedford MT: Arginine methylation at a glance. J Cell Sci 2007, 120:4243–4246.PubMedCrossRef 3. Bedford MT, Clarke SG: Protein arginine methylation in mammals: who, what, and why. Mol Cell 2009, 33:1–13.PubMedCrossRef 4. Krause CD, Yang ZH, Kim YS, Lee JH, Cook JR, Pestka S: Protein arginine methyltransferases: evolution and assessment of their pharmacological and therapeutic potential. Pharmacol Ther 2007, 113:50–87.PubMedCrossRef 5. Boisvert FM, Chénard CA, Richard S: Protein interfaces in signaling regulated by arginine methylation.