Figure 7.

RNAi of bloodstream form trypanosomes. (A) Growth curve of two independently derived clones in the absence (solid line) or presence (dashed line) of 1 μg ml⁻¹ tetracycline. The experiment was carried out in triplicate and bars denoting SD are shown. (B) Northern blots of total RNA prepared from parasites 12 h −/+ induction, electrophoresed on a 1% agarose gel and transferred onto nylon membrane. Duplicate blots were hybridized with ³²P-labeled ORFs of either GPI8 or MOB1. (C) Western blot analysis of whole cell lysate 24 h −/+ induction probed with α-GPI8, α-VSG 221, or α-aldolase. A Typhoon 8600 phosphoimager was used for detection and quantitation. (D) Cells, 1 × 10⁷, cultured for 24 h −/+ induction were lysed on ice in 25 μl 0.05% TX-100 with 1 mg ml⁻¹ pefabloc and 1 μg ml⁻¹ leupeptin and then incubated at 37°C for 30 min. Alternatively cells were lysed as above in the presence of 10 mM ZnCl₂ and maintained on ice for 5 min. Lysates were made up to 1 ml, after which they were centrifuged at 100,000 × g for 30 min at 4°C to yield P100 and S100 fractions. Samples were analyzed by Western blotting with α-VSG 221 or α-aldolase.