Amin-like proteinTable 1. Cont.AccessionTAPLOS A single | plosone.orgTATAHypotheticalPhosphorylation of Theileria annulata Schizont Surface Proteinsbut not completely, responsible for phosphorylating p104 during mitosis. In our existing perform, we identified three phosphorylated serines inside this fragment, and show that two of them (S601 and S607) are in reality highly phosphorylated through S-phase (Figure 7 and S7, Table S6). Collectively, the data from our present and previous function indicate that p104 is extensively phosphorylated, and that distinct residues are differentially phosphorylated inside a cell cycle-dependent manner. Thinking about the multiple protein-binding domains in p104, it will be of interest to additional investigate the putative interaction of p104 with other host cell proteins, along with the cell cycle-dependent alterations in phosphorylation described right here needs to be taken into account.Figure S3 Detection of p-Thr, p-Ser and p-Thr-Pro epitopes inuninfected bovine macrophages (BoMAC) throughout host cell interphase and mitosis (TIF) Synchronisation of TaC12 cells in S- or M-phase. A: Asynchronous TaC12 cells and cells incubated for 24 h in thymidine (S-phase) or 16 h in nocodazole (M-phase) were fixed in 80 ethanol and also the DNA content was labelled with propidium iodide before FACS analysis.3-Bromo-5-methylbenzonitrile Chemical name B: Lysates from TaC12 cells (unsynchronised, S-phase or M-phase) were analysed by Western blot employing anti-cyclin-A and anti-p-Histone H3 antibodies. As a loading handle anti-Theileria-HSP70 was utilised. C: Following synchronisation TaC12 cells had been fixed with four PFA and labelled with a polyclonal anti-schizont antibody and anti-p-Histone H3. DNA was visualised with DAPI. Merge: anti-p-Histone3 (green), anti-schizont (red) and DAPI (blue). Scale bar represents 10 mm. (TIF)Figure S4 Figure S5 Relative signal intensity following western blotting of TaC12 and schizont lysates with anti-p-Thr, p-Thr-Pro and p-Ser antibodies. Relative intensities were measured applying ImageJ, and correspond for the western blots shown in figure 5. (TIF) Figure S6 Parasite DNA replication happens as the host cell progresses by way of mitosis.5-Fluorobenzofuran-2-carboxylic acid structure A: TaC12 cells had been synchronised in S-phase with thymidine therapy, and released into fresh medium.PMID:24576999 BrdU (ten mM) was added for the culture two hours prior to evaluation at 6, eight, ten and 12 hours after thymidine release. Cells have been fixed with 4 PFA and BrdU incorporation into host (H) and parasite (P) nuclei was analysed by IFA. Quantification of cells that had incorporated no BrdU (H2/P2) are excluded from the graph for clarity. n = 702120 cells per time point. B: Representative pictures of cells at 6 hours (left) and 12 hours (ideal) post thymidine release are shown. The schizont is labelled green and BrdU is red. Scale bar represents 10 mm. (TIF) Figure S7 TaSP (TA17315) and p104 (TA08425) protein and phosphopeptide abundances. A: 3 phosphorylation-sites had been detected in TaSP. Two phosphorylated residues have been found with a greater abundance in S-phase (p,0.01). Data have been analysed with Progenesis. The max fold transform for every significant (p,005) paired scanning occasion for peptides that were differentially detected in between S- and M-phase are shown within a table (data extracted from table S6). The normalised peptide abundance for peptide SSSFSRINEDCC in S-phase and mitosis samples is presented as a histogram (consensus of all paired scanning events). B: 14 phospho-sites in p104 were detected (bold inside the proteinsequence). Two detected phosphorylated peptides (cor.