Screen for shRNAs that silence E1k and E2k expression. Eight shRNAs for each subunit were designed and constructed as described in Methods. (A) The structure of the shRNA-expressing constructs and an example for the shRNAs. (B) A Northern blot detecting silencing of the E1k subunit. Total RNA samples were prepared from cells 48 hours after transfection with the vectors shown in (A). Northern blot was carried out as described in Methods. The left five lanes are a dilution series of total RNA extracted from mouse NF1 cells transfected with a construct that expresses a scrambled shRNA (shR-Scr). The numbers at the bottom indicate the relative amount that was loaded in each lane. One hundred equals to 6 μg total RNA. In the right eight lanes, 6 μg total RNA extracted from cells transfected with each of the eight shRNAs against the E1k subunit were loaded. The probe detects the endogenous E1k mRNA. The numbers at the bottom indicate the relative density of the E1k band. (C) A Northern blot detecting silencing of the E2k subunit was done in the same way as in (B). The loading arrangement is the same as in (B) except that one hundred in the standard equals to 20 μg of total RNA. Twenty μg also is the amount of total RNA loaded in each sample lane.