Transcript-specific inhibition of translation by KRIPP1 RNAi. Sedimentation analysis of unedited CO1 mRNA (A) and 5′ edited cyb mRNA (B) in KRIPP1 knockdown cell line. Detergent-extracted lysate from approximately 2 × 108 parasites was separated in 10–30% glycerol gradient. RNA was extracted from each fraction and separated on 1.8% agarose-formaldehyde gels for Northern blotting.
C. Analysis of translation products in KRIPP1-depleted parasites. RNAi was induced for indicated periods of time and cells were labelled with EasyTag labelling mix (PerkinElmer Life Sciences) in isotonic buffer supplemented with 0.1 mg/ml of cycloheximide. Cells were collected by centrifugation, dissolved in SDS gel loading buffer and fractionated by 2D electrophoresis. Gels were stained with Coomassie Brilliant Blue R250 (inset panels) and exposed to X-ray film (large panels). Based on previous protein identifications (Nebohacova et al., 2004), major spots indicated by arrows represent CO1 and Cyb subunits.