Effect of DRBD18 RNAi on RBP10 mRNA and expression levels. (A) Example of cumulative growth curves of bloodstream forms with and without induction of DRBD18 RNAi. (B) DRBD18 and RBP10 protein levels after RNAi induction, corresponding to A. Ribosomal protein S9 is the loading control. Anti-RBP10 antibodies (Wurst et al. 2012) were used for detection of RBP10 and a nonspecific band (x). (C) RNA from the samples used in A was used to examine effects on RBP10 mRNA. The methylene blue-stained membrane is shown on the left, with RBP10 hybridization in the center. The membrane was then stripped and hybridized with a β-tubulin probe as a further control; *RBP10 signal that remained after stripping. (D) Visualization of RNA-seq results after 12 h tetracycline-mediated induction of DRBD18 RNAi, using the Integrated Genomics Viewer (Robinson et al. 2011; Thorvaldsdóttir et al. 2013). Three replicates are shown. Note that these results are shown on a linear scale whereas those in Figure 1A are on a log scale. Proposed full length mRNAs are shown below the tracks; positions of (U)12 or (U)6C(U)6 sequences are indicated in cyan but there are numerous other polypyrimidine tracts present. The cyan dotted line highlights loss of reads at the end of the RBP10 3′-UTR after 12 h DRBD18 RNAi. The read densities over the middle portion of the 3′-UTR are lower than the rest, probably because some of the reads will be aligned to the additional sequence copy in the TREU927 reference genome. Colored lines indicate read mismatches with the TREU927 reference genome.
