Figure 6.

Immunoblot analysis of enolase and eIF5A in P. falciparum schizont-stage parasites. (a) Two-dimensional Western blot of enolase. (b) One-dimensional Western blot of enolase. (c) Two-dimensional Western blot of eukaryotic initiation factor 5A (eIF5A). The upper panel shows the blotted nitrocellulose membrane after staining with a reversible protein dye (MemCode; Pierce), whereas the lower panel depicts the resulting immunoblot after detection with antibodies raised against a plant eIF5A. (d) One-dimensional Western blot of eIF5A. For each two-dimensional blot, 500 μg parasite protein (time point [TP]3, 42 hours post-invasion) was separated on a 13 cm isoelectric focusing (IEF) strip pH 4 to 7 (GE Healthcare) followed by an 11% polyacrylamide gel. Protein spots that could be matched on silver-stained gels were confirmed as P. falciparum enolase (panel a) and eIF5A (panel c) by tandem mass spectrometry and are indicated by arrowheads. For the one-dimensional blots, each lane was loaded with 10 μg parasite protein from the corresponding time point sample. eIF, eukaryotic initiation factor.