Figure 5. Generation of polydenylated histone mRNAs in Drosophila cells in the absence of dFLASH.

A. Western blot analysis of lysates from untreated D.Mel-2 cells (lane 1), or cells treated with the indicated dsRNAs, including dFLASH#1 (lane 2) that targets an upstream region in the dFLASH mRNA. The blot was probed with an anti-dsymplekin antibody (top panel) or anti-SLBP antibody (bottom panel). CR band in the bottom panel corresponds to a cross-reacting protein that is detected by the anti-SLBP antibody and serves as an internal loading control. B. Western blot analysis of lysates from untreated D.Mel-2 cells (lane 1) or cells treated with the dFLASH#1 dsRNA (lane 2). An aliquot of a nuclear extract (NE) from Drosophila Kc cells was analyzed in lane 1. The blot was probed with an anti-dFLASH antibody. CR band corresponds to a cross-reacting protein that is detected by the anti-dFLASH antibody in the D.Mel-2 whole cell lysates but not in the Kc NE. C. Northern blot analysis of total RNA isolated from D.Mel-2 cells tested in panel A and B. The blot was hybridized with ³²P-labeled probes directed against the Drosophila H3 (dH3) histone mRNA (top panel) and Drosophila 7SK RNA to control for equal loading (bottom panel). D. Western blot analysis of lysates from untreated D.Mel-2 cells (lane 1) or cells treated with the indicated dsRNAs, including the dFLASH#2 (lane 4) that targets a downstream region in the dFLASH mRNA. The blot was probed with an anti-dsymplekin antibody (top panel), anti-dSLBP antibody (middle panel), and anti-dFLASH antibody (bottom panel). E. Northern blot analysis of total RNA isolated from D.Mel-2 cells tested in panel D. The blot was hybridized with a ³²P-labeled probe directed against Drosophila H3 histone mRNA. An overexposure of the part of the film containing lanes 2–6 is shown to the right.