Figure 5. Ribozyme-mediated control of endogenous PfDHFR-TS expression.
(A) Knockdown of PfDHFR-TS expression in DHFR-TS-GFP_glmS integrant parasite clonal lines in response to GlcN. Parasitized erythrocytes expressing PfDHFR-TS-GFP were enumerated by flow cytometry based on the level of GFP fusion partner. Extra sum-of-squares F- test comparing individual curve fits with the null hypothesis that slope and EC50 are the same for both clone #1 and #2, P = 0.19. (B) Ribozyme-mediated knockdown of DHFR-TS-GFP protein is reversible. DHFR-TS-GFP_glmS integrant parasite clone #1 was cultured and treated with GlcN, and western immunoblotting to quantify DHFR-TS-GFP protein was performed as described in Fig. 3. Data are the mean from triplicate experiments and error bars represent S.E.M. (C) Representative images from Ponceau S staining of parasite lysates following electrophoresis and transfer to membrane, and chemiluminescent detection of GFP using specific antibodies. The pre-stained marker lane is marked M above the Ponceau S panel, and the sizes of two marker proteins are indicated.