Histological analysis Tumor samples were closely examined under the dissecting microscope and processed into paraffin

May 27, 2017

searches were performed against the SwissProt database to which was added the T. brucei subset of the TriTrypDB database v. 4.1 totaling 563,498 entries. For estimation of false discovery rates, the database was concatenated with a fully randomized set of sequence entries. Data were searched with mass tolerances of 20 ppm for parent and 0.6 Da for fragment ions. For database searching, peptide sequences were matched as tryptic peptides with no missed cleavages, and carbamidomethylated cysteines as a fixed modification. Variable modifications included oxidation of methionine, N-terminal pyroglutamate from glutamine, loss of methionine and N-terminal acetylation. Protein Prospector score parameters were set at a minimum protein score of 22, minimum peptide score of 15, and maximum expectation values of 0.01 for protein and 0.001 for peptide matches, resulting in a protein false discovery rate of 0.4%. Protein identification results from specific TAP experiments were reported with a spectral count as an approximation of protein abundance, along with percent sequence coverage and an expectation value for the probability of protein identification. Flow Cytometry Flow cytometry was performed on propidium iodide -stained cells to determine the DNA contents of the cells. At each 24 hr interval after RNAi-induction, cells were fixed, stained and processed for fluorescence activated cell sorting scan analysis according to the previously 22431203 established protocol. The DNA peaks of PI-stained cells were analyzed with the FACScan analytical flow cytometer. FL2-A DNA peaks were calculated using CellQuest software. Cells were also harvested, washed once with PBS, attached to Poly-LLysine coated cover slips and mounted in vectashield medium with DAPI for microscopic examination of SR 2516 nucleus / kinetoplast configurations. Percentages of cells with different N&K configurations in each sample were determined by counting at least 200 cells with an epifluorescence microscope. Epitope Tagging of Endogenous Proteins in Procyclicform T. brucei The 39-terminal fragment of CDC27, AP1 and AP2 genes were amplified by PCR and cloned into the pC-PTP-NEO plasmid. The three resulting DNA constructs, pC.CDC27.PTP, pC.AP1.PTP and pC.AP2.PTP were linearized with AvaI, BbsI and XhoI, respectively, and transfected into the strain 427 cells by electroporation. Stable clonal transfectants were selected under 40 mg ml21 G418. For 3HA-epitope tagging of the endogenous mitotic cycB2/cyc6 gene, the C-terminal fragment of the gene was amplified from the genomic DNA using gene specific primers and cloned into the pC.3HA.Bla plasmid, which is a modified version of the endogenous targeting The APC/C of Trypanosoma brucei plasmid pC.PTP.NEO, and was transfected into either 16483784 29-13 procyclic cells or 29-13 cells with a stably maintained AP2 RNAi plasmid. Transfectant selection was carried out under 10 ug ml21 Blasticidin. Western Blotting Cells were harvested, washed twice in phosphate-buffered saline, lysed by sonication in SDA-PAGE laemmli sample buffer and cleared by a brief centrifugation. Samples were fractionated on SDS-PAGE and transferred onto PolyVinylidene DiFluoride membrane. After blocking in TBST containing 5% skim milk, the immuno-blot membrane was probed with primary anti-Prot C HPC4 monoclonal antibodies and stained with the secondary anti-mouse IgGHRP conjugate. Immunoprecipitation Cells were harvested, washed once in PBS and the cells extracted in the lysis buffer for 30 min