B) In vivo interaction between TbLpn and TbPRMT1.

TbLpn was immunoprecipitated from PF T. brucei cytosolic extracts using anti-TbLpn polyclonal Protein Tyrosine Kinase inhibitor antibodies as described under Material and Methods. As a negative control, the cytosolic extract ERK inhibitor was incubated in the absence of antibodies. Proteins present in the starting cytosolic fraction (C), as well as the bound (B) and unbound fractions (U) were separated on a 10% polyacrylamide gel and transferred to PVDF. The presence of TbLpn in the immune complexes was assessed by probing the membrane with anti-TbLpn polyclonal antibodies (1:1,000), followed by goat anti-rabbit IgGs. The presence of TbPRMT1 in the immune complexes was detected by probing the blot with anti-TbPRMT1 polyclonal antibodies (1:1,000), followed by goat anti-rabbit IgGs. Signals were detected using chemiluminescence. In order to examine the interaction between TbPRMT1 and TbLpn in vivo, we performed a co-immunoprecipitation. As shown above, TbLpn is located in the cytosol of the parasite. For this reason, TbLpn was immunoprecipitated from PF T. brucei cytosolic extracts using purified polyclonal anti-TbLpn

antibodies. Proteins that were immunoprecipitated along with TbLpn were separated by electrophoresis and transferred onto PVDF. The presence of TPRMT1 in association with TbLpn was determined by using purified polyclonal anti-TbPRMT1 antibodies to probe the membrane by western hybridization. The results shown in Figure 4B clearly show that a band of approximately the size of TbPRMT1 (38.9 kDa) co-precipitates exclusively with TbLpn, and is not MK5108 manufacturer present in the negative control. TbLpn is methylated in vivo The physical association of TbPRMT1 with TbLpn suggests that TbLpn may serve as a substrate for methylation by TbPRMT1. In support of this hypothesis, several arginine residues throughout the TbLpn sequence are located within preferred motifs for methylation, such as RG or RXR. To evaluate whether TbLpn is methylated in vivo, an immunoprecipitation

was performed from PF T. brucei cytosolic extracts using purified anti-TbLpn polyclonal antibodies. The presence of methylated arginine residues was Ribonucleotide reductase then determined by western hybridization using anti-mRG polyclonal antibodies. These antibodies were raised against a peptide containing 7 asymmetric dimethylarginine residues alternating with 8 glycine residues. This motif is found most prevalently among verified dimethylarginine- containing proteins. The antibodies have been shown to specifically recognize methylated arginine residues [52]. Using these antibodies to probe the blot, a protein band was observed at 85 kDa, which is the predicted size of TbLpn, in the bound but not the unbound fraction (Figure 5). This clearly indicates that native TbLpn contains methylated arginine residues.