Figure 5

Northern blots of small RNAs extracted from Igl and PATMK transfectants. To test if the U6 promoter was driving hairpin expression, shRNA transfectants (PATMK (3552–3580), PATMK (2273–2301), PATMK (3552–3580 scrambled) [39], Igl (1198–1226), Igl (2412–2440), and Igl (2777–2805) were selected with 30 μg/ml hygromycin for 48 hours before harvesting. HM1:IMSS non-transfected amebae were included as negative controls. Small RNAs were extracted using the mirVana™ miRNA Isolation Kit (Ambion) (Applied Biosystems/Ambion, Austin, TX, USA). Fifty μg small RNA were loaded per lane on a 12% denaturing acrylamide gel and transferred to membrane. rRNA bands were analyzed to ensure equal RNA loading. Oligo probes matching to the sense and antisense strands of the hairpins were end-labeled with ³²P and were hybridized with each corresponding sample blot overnight at 37°C overnight, washed with low and medium stringency conditions, and exposed overnight to film. Note the two product sizes, which may correspond to the unprocessed hairpin (~60–70 nucleotides) (blue arrows) and the processed siRNA products (~30 nucleotides) (red arrows).