Erature to ensure complete binding. Subsequently, the peptide solutions were removed from the wells and the entire plate was washed three times 3 minutes each in buffer A to remove any unbound peptides. The plate was then incubated with GST14-3-3I and Dimethylenastron web GST-14-3-3II [1.0 mg/100 ml per well], GSTHP1CD and GST [0.50 mg/100 ml per well] diluted in buffer A at appropriate wells containing different peptides. All the proteins of same dilutions as above were also put into wells that were streptavidin coated but not incubated with peptides, to determine if any of the proteins bound directly to streptavidin. After incubating at room temperature for 2 hours, the protein solutions were removed, and the plate was washed three times 3 minutes each in buffer A. The plate was next incubated at room temperature for 1 hour with anti-GST-HRP antibody diluted in buffer A [1:5000], following which the wells were washed three times 3 minutes each in buffer A. One streptavidin coated well which was not incubated with any peptides or proteins, was incubated with anti-GST-HRP antibody to determine any direct interaction between streptavidin and anti-GST-HRP antibody. All the wells were then fluorogenically developed with Amplex Ultra Red reagent (Invitrogen; A36006) as per manufacturer’s recommendation and quantified using a Safas spectrophotometer. All experiments were performed in triplicate.In silico modelling of Pf14-3-3 proteinsAmino acid sequences of Pf14-3-3I and Pf14-3-3II were submitted to the I-TASSER server for structural prediction [29,30]. Protein structures were visualized using MacPyMol version 0.99rc6. Sequence alignments of Pf14-3-3I and Pf14-33II to 14-3-3 proteins from human (NP_003397), Nicotiana tobaccum (P93343), and Cryptosporidium parvum (cdg3_1290) were performed using ClustalW2 and visualized using BOXSHADE.ELISA-based protein binding assayBinding of GST-14-3-3I and GST-14-3-3II proteins to purified parasite histones and phospho-modified histone peptides was checked in ELISA assay. Maxisorp NUNC ELISA plates were used for all the assays. Buffer A (50 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM DTT, 100 mM NaF, 0.005 Tween-20, 1 BSA, and 10 Glycerol) was used as binding and washing buffer unless mentioned otherwise.Purified parasite histones and 14-3-3 protein binding assay. Core histones were purified from unsynchronized 3D7 parasites (see above) and diluted in Tris pH 8.0 at 0.40 mg/100 mlResults Development of improved acid and high-salt purification methods for P. falciparum histone phosphoprotein analysisPrevious studies on histone modifications in P. falciparum [8,23] did not detect any phosphorylation marks on histones (reviewed in [31]). Most of these studies relied on traditional acid extraction method to partially purify parasite histones and 1527786 did not include affinity enrichment of the 871361-88-5 phosphopeptides [17,21,32,33]. We developed two methods to yield purer histones while maintaining phospho-marks and included a phosphopeptide enrichment step in our study (Figure 1). A typical purified histone sample obtained by acid extraction is shown in Figure 2A. To confirm that the samples had retained their phospho-PTMs, we probed our purified histone samples with commercially available antibodies against the H3S10ph, H3T11ph, and H3S28ph modifications of histone H3, which is highly conserved between P. falciparum and human. Western blot analysis performed with these antibodies on histones extracted by either acid (Figure.Erature to ensure complete binding. Subsequently, the peptide solutions were removed from the wells and the entire plate was washed three times 3 minutes each in buffer A to remove any unbound peptides. The plate was then incubated with GST14-3-3I and GST-14-3-3II [1.0 mg/100 ml per well], GSTHP1CD and GST [0.50 mg/100 ml per well] diluted in buffer A at appropriate wells containing different peptides. All the proteins of same dilutions as above were also put into wells that were streptavidin coated but not incubated with peptides, to determine if any of the proteins bound directly to streptavidin. After incubating at room temperature for 2 hours, the protein solutions were removed, and the plate was washed three times 3 minutes each in buffer A. The plate was next incubated at room temperature for 1 hour with anti-GST-HRP antibody diluted in buffer A [1:5000], following which the wells were washed three times 3 minutes each in buffer A. One streptavidin coated well which was not incubated with any peptides or proteins, was incubated with anti-GST-HRP antibody to determine any direct interaction between streptavidin and anti-GST-HRP antibody. All the wells were then fluorogenically developed with Amplex Ultra Red reagent (Invitrogen; A36006) as per manufacturer’s recommendation and quantified using a Safas spectrophotometer. All experiments were performed in triplicate.In silico modelling of Pf14-3-3 proteinsAmino acid sequences of Pf14-3-3I and Pf14-3-3II were submitted to the I-TASSER server for structural prediction [29,30]. Protein structures were visualized using MacPyMol version 0.99rc6. Sequence alignments of Pf14-3-3I and Pf14-33II to 14-3-3 proteins from human (NP_003397), Nicotiana tobaccum (P93343), and Cryptosporidium parvum (cdg3_1290) were performed using ClustalW2 and visualized using BOXSHADE.ELISA-based protein binding assayBinding of GST-14-3-3I and GST-14-3-3II proteins to purified parasite histones and phospho-modified histone peptides was checked in ELISA assay. Maxisorp NUNC ELISA plates were used for all the assays. Buffer A (50 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM DTT, 100 mM NaF, 0.005 Tween-20, 1 BSA, and 10 Glycerol) was used as binding and washing buffer unless mentioned otherwise.Purified parasite histones and 14-3-3 protein binding assay. Core histones were purified from unsynchronized 3D7 parasites (see above) and diluted in Tris pH 8.0 at 0.40 mg/100 mlResults Development of improved acid and high-salt purification methods for P. falciparum histone phosphoprotein analysisPrevious studies on histone modifications in P. falciparum [8,23] did not detect any phosphorylation marks on histones (reviewed in [31]). Most of these studies relied on traditional acid extraction method to partially purify parasite histones and 1527786 did not include affinity enrichment of the phosphopeptides [17,21,32,33]. We developed two methods to yield purer histones while maintaining phospho-marks and included a phosphopeptide enrichment step in our study (Figure 1). A typical purified histone sample obtained by acid extraction is shown in Figure 2A. To confirm that the samples had retained their phospho-PTMs, we probed our purified histone samples with commercially available antibodies against the H3S10ph, H3T11ph, and H3S28ph modifications of histone H3, which is highly conserved between P. falciparum and human. Western blot analysis performed with these antibodies on histones extracted by either acid (Figure.
