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ALMUDENA CROOKE Y COLS. AN. R. ACAD. NAC. FARM.

corresponding to the theoretical molecular mass of the deduced
protein sequence and the 73 kDa band with 2 other subforms in the
mature parasites.

Compared to the control, gene silencing by dsiRNA reduced
G6PD-6PGL immunoreactivity as observed in cultures at 3 and a
24 h (Fig. 3B). Thus, as earlier as 3 h, a 54% and 69% reductions in
the 73 kDa and 107 kDa bands, respectively, were observed (Fig. 3C).
After 24 h, this effect started to diminish, and only 14% and 19%
decreases in the two bands, respectively, were observed (Fig. 3C).
These data confirm previous findings that silencing through dsRNA-
G6PD instantly took place after 3 h of electroporation at the ring
stage, with normal levels gradually restored at the trophozoite stage,
24 h post-transfection (18).

w t wt

FIGURE 3. Effect of dsRNA on parasite protein levels. (A) Immunoblot analysis
of wild-type synchronized 3D7 rings and trophozoites. The G6PD-6PGL protein
band pattern immunodetected during the intraerythrocyte cycle of P. falciparum

includes the 107, 73, 69 and 53 kDa bands labelled with arrows. (B) Immunoblot
analysis of synchronized wild-type (w) and dsRNA-G6PD transfected (t)

parasites (10 µg of total protein per lane) at 3 and 24 h after electroporation.
(C) Immunoblot quantification of synchronized wild-type and dsRNA-G6PD

parasites. The 107 and 73 kDa bands signal detected were quantified by
densitometry using a Fluor-S MultiImager and Quantity One quantitation
software (Bio-Rad). Value for PfG6PD-6PGL in dsRNA-G6PD parasites is
expressed as a percentage of control levels in wild-type parasites transfected
with water. The positions of molecular mass standards are indicated (kD).

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