Figure 3.—

Effects of RNA interference (RNAi) in living flies. (A) The 650-bp D-DDB1 double-strand RNAs were expressed using the Act5C-GAL4 driver and Northern hybridization analysis was performed for total RNAs from third instar larvae carrying a single copy of Act5C-GAL4 (lane 1, +/+;Act5C-GAL4/+) or single copies of Act5C-GAL4 and UAS-D-DDB1(650)-dsRNA (lane 2, UAS-D-DDB1(650)-dsRNA/+;Act5C-GAL4/+). Total RNAs were separated on a 1.2% agarose-formaldehyde gel, transferred onto a nylon membrane, and probed with a random-primed ³²P-labeled D-DDB1 ORF (1774–3420 bp). The blot was reprobed for the RP49 message as a loading control. (B) The transcripts of D-DDB1 were reduced to 7.3% by RNAi compared with the amount of RP49 transcripts. The amount of PCNA was not changed after inducing the D-DDB1 gene silencing. (C) D-DDB1, RPA30, and α-tubulin in an Act25-GAL4/+ fly (lane 1) and an Act25-GAL4/UAS-D-DDB1(650)-dsRNA fly (lane 2) were determined by immunoblotting. The protein levels of D-DDB1 were also reduced by RNAi.