Plasmid dna

All primers used are listed in Table 1, and all plasmids were sequenced and analyzed before transfection. For the PfSortilin-3HA-tagged line, a fragment of 1 kb upstream of the stop codon of the PfSortilin gene was amplified from P. falciparum 3D7 cDNA and cloned into the BglII-PstI site of the pHA3 vector (32 (link)). Wild-type (WT) P. falciparum 3D7 parasites were transfected with the PfSortilin-3HA plasmid, and integrants were selected and cloned as described previously (33 (link)). Briefly, P. falciparum 3D7 parasites were transfected with 100 μg of purified plasmid DNA (Promega). Integrated parasites were selected using 20 nM WR99210 (Jacobus Pharma).
The RAMA fragments were PCR amplified from P. falciparum cDNA and cloned into the MluI-SpeI sites in the pTET-MSP2p-SP-mCherry which allows schizont-stage expression and entry into the secretory pathway. The transfectants were kept on 0.5 µg/ml anhydrotetracycline to prevent transgene expression.
To generate the green fluorescent protein (GFP)-Rab7 constructs, Rab6 was removed from pARL-GFP-Rab6 (34 (link)) and replaced by full-length Rab7 amplified from cDNA and digested with AvrII-XhoI.

Gold(III) chloride trihydrate, branched polyethylenimine (25 kDa), sodium borohydrite, 11-mercaptoundecanoic acid, N-hydroxysuccinimide, and fetal bovine serum (FBS) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Penicillin-streptomycin solution, trypsin-EDTA solution, and alpha-MEM, were obtained from Life Technologies (GIBCO, Grand Island, NY, USA). Lipofectamine 2000 and LysoTracker Green DND-L7526 were purchased from Invitrogen (Carlsbad, CA, USA). Plasmid DNA, pGL3-Control, and the Luc assay kit were obtained from Promega Corp (Madison, WI, USA). The bicinchoninic acid (BCA) protein assay system was obtained from Thermo Scientific (Rockford, IL, USA). The antibodies used for immunoblotting were against EGFP (Cell Signaling Technology, Beverly, MA, USA), C/EBPβ (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and beta-actin (Abcam, Cambridge, MA, USA).

To create the plasmid used for the integration of the RFA at the 3'end of the PfPrp2 gene by single crossover, a PCR fragment containing nucleotides 2819 to 3774 of PfPrp2 without a stop codon was cloned into the Xho1-AvrII sites of the pGDB vector [17 (link)]. Because wild-type P. falciparum is sensitive to trimethoprim, the DHFRdd system requires the use of parasite strains resistant to this antifolate. The system was developed with a PfPlasmepsin 1 knock out strain (ΔPM1) (PfPM1 is a non essential gene) expressing hDHFR, rendering the parasites resistant to TMP[17 (link)] so we decided to use this same strain for our studies. Parasites were transfected, and integrants were selected as described previously[48 (link)]. Briefly, P. falciparum 3D7 ΔPM1 parasites [49 (link)] were transfected with 100 ug of purified plasmid DNA (Promega). Positive selection for transfectants was achieved using 2.5 mg/ml BSD (Sigma-Aldrich) and 5 uM TMP (Sigma-Aldrich). Integration was monitored by Southern blots according to standard procedures.