FIGURE 3.

Antisense sRNAs are detected for EhSTIRP in G3:STIRP knockdown parasites. A, top, location of the strand-specific oligonucleotide probes on the EhSTIRP1 gene (arrows in black are probes with Northern blots pictured in the figure(s); arrows in gray are the probes without Northern blots pictured but mentioned under “Results” as data not shown). Sense probes for detecting antisense sRNAs are designed for three regions: for the 5′-trigger region (SS1 and SS624), for the 3′-trigger region (SS6188, SS6342, SS6501, and SS6801), and for the region outside the triggers (SS1028 and SS2520). The trigger region cloned into psAP-2 is shown below the EhSTIRP1 schematic; for 5′STIRP, it is between positions 1 and 984; for 3′STIRP, it is between positions 6188 to 6966. Bottom, Northern blot detects antisense sRNAs for EhSTIRP in G3:STIRP knockdown parasites. A Northern blot (loaded with 50 μg of sRNA-enriched sample from E. histolytica trophozoites of G3, G3:5′STIRP, and G3:3′STIRP) were probed with oligonucleotide probes shown in the top to detect antisense sRNAs. The blot was sequentially probed, stripped, and reprobed; probes used are shown below each blot. Methylene blue staining is shown as a loading control. B, Northern blot analysis shows gene silencing of EhSTIRP transcript. A Northern blot loaded with 15 μg of total RNA for each sample was probed with PCR probes for EhSTIRP, ap-a, and actin (as a loading control). The down-regulation of EhSTIRP is shown for G3:3′STIRP and G3:5′STIRP. Due to the high degree of similarity between genes in the EhSTIRP gene family, it is unclear whether low signal in the G3:5′STIRP lane represents transcript from EhSTIRP1 or if there is cross-hybridization with other EhSTIRP gene family members.