Fig. 8: PfSTART1 expression and processing. | Nature Communications

Fig. 8: PfSTART1 expression and processing.

From: Aryl amino acetamides prevent Plasmodium falciparum ring development via targeting the lipid-transfer protein PfSTART1

Fig. 8

A Western blot of SLI-WT parasites along the 48 h erythrocytic cycle. A saponin-lysis was performed on tightly synchronised SLI-WT young rings, older rings, young trophozoites, older trophozoites, and schizonts. B Densitometry of PfSTART1 over three biological replicates (normalised by the corresponding PfHSP70-1 signal; mean +/− SD). C Synchronous 3D7 trophozoites were magnet-purified, and whole cells harvested at ~40 hpi (hour post invasion), ~44 hpi and ~48 hpi (this last sample was treated with 10 µM E64 to prevent egress). PEXEL-cleaved PfSTART1 and two further processed forms are indicated by solid and empty arrows respectively. D To localise PfSTART1, Percoll-purified 3D7 schizonts were sequentially lysed with equinatoxin II (EqtII), saponin (Sap) and Triton-X100 (TX-100), and the supernatants (SN) were collected to harvest proteins localising in the red blood cell (RBC) cytosol, the parasitophorous vacuole (PV) and the parasite, respectively. PfGBP130 is a protein known to be exported into the RBC cytosol; PfSERA5 localises to the PV in schizonts; PfHSP70-1 is a parasite cytosolic protein. E To determine the solubility of PfSTART1, saponin-lysed 3D7 schizonts were sequentially lysed in PBS (with five freeze-thaw cycles) and sodium carbonate. The supernatant resulting from the PBS lysis was collected (PBS soluble), as well as the supernatant and the pellet of the carbonate lysis (Carbonate soluble and Carbonate insoluble respectively). Controls are the soluble protein PfHSP70-1, the membrane-associated protein PfHSP101, and the integral transmembrane protein PfEXP2. F Proteinase K protection assay was conducted on Percoll-purified 3D7 schizonts. Schizonts were first lysed in EqtII, the supernatant (SN) of which was collected. The remaining parasite and PV were either incubated in PBS (no lysis), saponin (PVM-lysis), or TX-100 (lysis of all membrane) with or without proteinase K. PfGBP130 is RBC cytosolic protein; PfPTEX150 is a PV protein; PfActin-1 is a parasite cytosolic protein. Note that another replicate for each of these experiments is shown in Fig S8. Source data are provided as a Source Data file.

Back to article page