ion, which is a population enriched with G1 cells up to 80% shown in flow cytometry. The cells were then synchronized with 0.3 mM hydroxyurea by an established procedure resulting in 100% of the cells arrested in late S-phase. They were then released from hydroxyurea and allowed to grow synchronously for 2.5 hrs when 65% of the population was found to have reached the metaphase and 15% in the anaphase in flow cytometry and immunofluorescence assays using chromosome passenger complex 1 protein as the marker . When the time period of synchronous growth was extended to 4.5 hrs, 6570% of the cells were in the anaphase, whereas the rest of the cells had apparently gone beyond cytokinesis and reached the G1 phase. The method thus enabled us to prepare large batches of procyclic-form T. brucei cells enriched in G1-phase, S-phase, metaphase and anaphase, respectively, and subject them to the TAP. The results from SDS-PAGE of the purified APC1-PTP protein complexes from the cells enriched in different phases of the cell cycle are presented in TAP of the APC1-PTP Protein Complex from T. brucei In order to identify the intact APC/C core from T. brucei, the potential scaffold protein subunit APC1 in the complex LC-MS/MS Analyses of T. brucei APC/C Derived from Different Phases of Cell Cycle Samples of the APC1-PTP fusion protein complex purified from duplicate or triplicate cell samples, each enriched in different The APC/C of Trypanosoma brucei 5 The APC/C of Trypanosoma brucei phases of the cell cycle, were fractionated in SDS-PAGE. Individual protein bands in the gels were sliced out, diced and digested with trypsin by the well-established procedures. As a control, lysate of 
427 cells transfected with empty pC.PTP plasmid was purified by the same procedure and individual protein bands were sliced from the SDS-PAGE and processed for trypsin digest as 12504917 mentioned above. Each digest was then fractionated with LC, and subjected to MS/MS analysis for peptide identification. The results in protein identities versus the individual protein bands in a SDS-PAGE gel, are represented in The APC/C of Trypanosoma brucei adequate numbers of unique peptides and sufficient percentages of coverage in the two tables with but one exception with APC11; data on this protein are apparently missing in the samples from G1-phase and anaphase enriched cells. This is most likely attributed to the relatively low molecular weight of APC11 that facilitates diffusion of the protein from the gel during SDS-PAGE. This small protein is thus still considered an integral component of T. brucei APC/C. To further verify that there is no significant fluctuation of the intracellular levels of APC/C subunits during cell cycle progression, APC1, CDC27, AP1 and AP2 were each tagged with PTP at the C-termini, integrated into chromosomes via homologous recombination and expressed in transfected cells as previously described. The transfected cells were then synchronized with hydroxyurea into late S-phase and released for synchronized growth for 8 10604535 hrs, which covers the entire cell cycle of T. brucei. Hourly samples were taken during the incubation and examined on Western blots. The results, presented in suggesting MSC1936369B web limited fluctuation in the level of APC/C subunits during cell cycle progression of procyclic-form T. brucei. Tentative Identification of AP1 as APC4 In NCBI BLAST analysis, the sequence of AP1 was successfully aligned with the protein fragments from the core APC/C subunit APC4 from s
