I-H3S28 antibody, after being incubated with or without phosphatase. The

I-H3S28 antibody, after being incubated with or without phosphatase. The phosphorylation signal was greatly reduced after phosphatase treatment (data not shown). The control peptide H3K9me3 was similarly incubated with or without phosphatase and binding of GST-HP1CD to the peptide after the treatment was determined. No significant reduction in binding of GST-HP1CD to theHistone Phosphorylation in P. falciparumTable 2. List of biotinylated H3-peptides used for ELISA binding assay.synthetic peptide name H31?0 H3S10ph H3S10phK14ac H3K9me31?0 H321?0 H3S28ph H3S28phS32phhistone H3 amino acid sequence ARTKQTARKSTAGKAPRKQL-K(biotin) ARTKQTARKS(ph)TAGKAPRKQL-K(biotin) ARTKQTARKS(ph)TAGK(ac)APRKQL-K(biotin) ARTKQTARK(tri-methylated)STAGKAPRKQL-K(biotin) ASKAARKSAPISAGIKKPHR-K(biotin) ASKAARKS(ph)APISAGIKKPHR-K(biotin) ASKAARKS(ph)APIS(ph)AGIKKPHR-K(biotin)doi:10.1371/journal.pone.0053179.tH3K9me3 peptide being observed after phosphatase treatment (data not shown).14-3-3I is present in both cytoplasmic and nuclear compartments of the intra-erythrocytic parasitePrevious studies identified 14-3-3 protein in P. berghei, P. knowlesii and P. falciparum in total extracts [42?4], but no data on subcellular localization are available. An anti-14-3-3I antibody was generated against the full-length GST fusion protein and was used in western blot to probe cytoplasmic and nuclear extracts from unsynchronised 3D7 parasites. A single band corresponding to expected size of the protein (,30 kDa) was observed in both fractions, whereas it did not recognize mammalian isoforms present in human erythrocytes, indicating the antibody was specific for Pf14-3-3I and the protein was present in both cytoplasmic and nuclear compartments of the parasite (Figure 5A). Subsequently, the same antibody was used in IFA to visualize the location of the protein throughout the life cycle of intra-erythrocytic parasite (Figure 5B). An IFA signal was observed in cytoplasmic compartment and overlapping with the nuclear signal at all asexual stages of the parasite, which is compatible with the protein immunoblot results. Since 14-3-3 buy 79831-76-8 proteins have been reported to be able to bind a multitude of functionally variant proteins and not only histones in other eukaryotic organisms [35,45,46], the 24195657 presence of the protein in different cellular compartments is consistent with its probable pleiotropic role of these proteins in P. falciparum.In silico structural analysis of Pf14-3-3 proteins revealed conservation of residues responsible for phosphoserine bindingPf14-3-3I and Pf14-3-3II amino acid sequences were submitted to the I-TASSER server for protein structure prediction [29,30]. The ITI 007 returned sequence alignments and structural analogues were exclusively 14-3-3 proteins from other organisms, including human, tobacco, and Cryptosporidium parvum. The I-TASSER server also predicted five structural models for each of the two P. falciparum 14-3-3 proteins. The highest scoring models of Pf14-33I and Pf14-3-3II are displayed alongside the structure of human 14-3-3 zeta co-crystallized with phosphorylated histone (H3S10ph) peptide in Figure 6A. As predicted from the primary protein sequence alignments, all the Pf14-3-3 structural models revealed the lysine, arginine, and tyrosine amino acid side chains involved in phosphoserine recognition to be positioned similarly to those in the solved 14-3-3 protein structures from model organisms [36,37] (Figure 6B). The backbone chains of all ten structural mode.I-H3S28 antibody, after being incubated with or without phosphatase. The phosphorylation signal was greatly reduced after phosphatase treatment (data not shown). The control peptide H3K9me3 was similarly incubated with or without phosphatase and binding of GST-HP1CD to the peptide after the treatment was determined. No significant reduction in binding of GST-HP1CD to theHistone Phosphorylation in P. falciparumTable 2. List of biotinylated H3-peptides used for ELISA binding assay.synthetic peptide name H31?0 H3S10ph H3S10phK14ac H3K9me31?0 H321?0 H3S28ph H3S28phS32phhistone H3 amino acid sequence ARTKQTARKSTAGKAPRKQL-K(biotin) ARTKQTARKS(ph)TAGKAPRKQL-K(biotin) ARTKQTARKS(ph)TAGK(ac)APRKQL-K(biotin) ARTKQTARK(tri-methylated)STAGKAPRKQL-K(biotin) ASKAARKSAPISAGIKKPHR-K(biotin) ASKAARKS(ph)APISAGIKKPHR-K(biotin) ASKAARKS(ph)APIS(ph)AGIKKPHR-K(biotin)doi:10.1371/journal.pone.0053179.tH3K9me3 peptide being observed after phosphatase treatment (data not shown).14-3-3I is present in both cytoplasmic and nuclear compartments of the intra-erythrocytic parasitePrevious studies identified 14-3-3 protein in P. berghei, P. knowlesii and P. falciparum in total extracts [42?4], but no data on subcellular localization are available. An anti-14-3-3I antibody was generated against the full-length GST fusion protein and was used in western blot to probe cytoplasmic and nuclear extracts from unsynchronised 3D7 parasites. A single band corresponding to expected size of the protein (,30 kDa) was observed in both fractions, whereas it did not recognize mammalian isoforms present in human erythrocytes, indicating the antibody was specific for Pf14-3-3I and the protein was present in both cytoplasmic and nuclear compartments of the parasite (Figure 5A). Subsequently, the same antibody was used in IFA to visualize the location of the protein throughout the life cycle of intra-erythrocytic parasite (Figure 5B). An IFA signal was observed in cytoplasmic compartment and overlapping with the nuclear signal at all asexual stages of the parasite, which is compatible with the protein immunoblot results. Since 14-3-3 proteins have been reported to be able to bind a multitude of functionally variant proteins and not only histones in other eukaryotic organisms [35,45,46], the 24195657 presence of the protein in different cellular compartments is consistent with its probable pleiotropic role of these proteins in P. falciparum.In silico structural analysis of Pf14-3-3 proteins revealed conservation of residues responsible for phosphoserine bindingPf14-3-3I and Pf14-3-3II amino acid sequences were submitted to the I-TASSER server for protein structure prediction [29,30]. The returned sequence alignments and structural analogues were exclusively 14-3-3 proteins from other organisms, including human, tobacco, and Cryptosporidium parvum. The I-TASSER server also predicted five structural models for each of the two P. falciparum 14-3-3 proteins. The highest scoring models of Pf14-33I and Pf14-3-3II are displayed alongside the structure of human 14-3-3 zeta co-crystallized with phosphorylated histone (H3S10ph) peptide in Figure 6A. As predicted from the primary protein sequence alignments, all the Pf14-3-3 structural models revealed the lysine, arginine, and tyrosine amino acid side chains involved in phosphoserine recognition to be positioned similarly to those in the solved 14-3-3 protein structures from model organisms [36,37] (Figure 6B). The backbone chains of all ten structural mode.