Among these few RBPs, the members of the STAR proteins family, and in particular SAM68, stand out

restriction sites, creating His-tagged PfSR1 similar to the His-tagged SRSF1 previously described. The synthetic pfsr1 fused to a T7 tag was cloned into pcDNA3 plasmid using XhoI and KpnI restriction sites creating a similar plasmid to the pCDNA3-SRSF1 previously described. PfSR1 was cloned into the expression vector pHBIRH previously PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19815606 described and fused to a myc epitope tag to generate pHBISR1myc. In order to purify the PfSR1-His protein, cells were transfected using linear polyethylenimine. A transfection mix consisting of 1 mg plasmid DNA, 2 mg PEI and 50 ml of JMEM was prepared for every 1 ml of cell culture to be transfected. The mix was incubated at room temperature for 1015 min and was then added to the cell culture. Forty-eight hours LY3039478 web post-transfection cells were collected and analyzed. Parasite transfections Parasites were transfected as previously described. Briefly, 0.2 cm electroporation cuvettes were loaded with 0.175 ml of erythrocytes and $100 mg of plasmid DNA in incomplete cytomix solution. Stable transfectants were initially selected on 2 mg/ml blasticidin-S-hydrochloride. In order to obtain parasites carrying high plasmid copy numbers, these cultures were then subjected to 6 mg/ml blasticidin. Genomic DNA extraction, RNA extraction and cDNA synthesis Genomic DNA was extracted as described. Briefly, iRBCs were pelleted and lysed with Saponin. RBC-free parasites from 20 ml cultures were then pelleted, washed with phosphate buffered saline, and taken up in 200 ml TSE buffer, 40 ml of 10% sodium dodecyl sulfate and 20 ml 6 M NaClO4. This suspension was rocked for 12 h and genomic DNA was extracted with phenol/chloroform. The resulting DNA was taken up in 100 ml dH2O. RNA extraction and cDNA synthesis were performed as described. Briefly, RNA was extracted from synchronized parasite cultures at 36 h post-invasion. RNA was extracted with the TRIZOL LS Reagent as described and purified on PureLink column according to manufacturer’s protocol. Isolated RNA was then treated with recombinant Deoxyribonuclease I to degrade contaminating gDNA. cDNA synthesis was performed from 750 ng total RNA with Primescript RT reagent with oligo dT as described by the manufacturer. Real-time reverse transcriptaseqPCR Transcript copy numbers were determined using the formula 2CT as described in the Applied Biosystems User Bulletin 2 using NF54 gDNA as the calibrator. Specifically, relative copy number was calculated as two exponential negative ). Relative copy numbers of pfsr1 and renilla luciferase were calculated comparing to the expression of the housekeeping gene arginyltRNA synthetase. All reverse transcriptase qPCR assays were performed at least in duplicate for each template with no apparent differences, and each experiment was completed three times in its entirety, again with no significant differences. The housekeeping and other control primers used for RTqPCR were previously published and arginyltRNA synthetase in ). Ectopic expression of pfsr1 was measured using primers designated to specifically amplify the fusion of pfsr1 and the myc tag. Protein purification 293T cells were transfected with human pTT3-SRSF1-His or pTT3-PfSR1-His and harvested 48 h after transfection. Cells were re-suspended in lysis buffer and centrifuged after sonication to remove insoluble material. Most cellular proteins were precipitated by using ammonium sulfate at 40% saturation at 4 C and were separated by centrifugation. His-tagged proteins were purified fr