Figure 7.

The CAF1–CCR4–NOT complex deadenylates mRNAs associated with CUP. (A,B) S2 cells were treated with the indicated dsRNAs on days 0 and 4. Control cells were treated with an unrelated dsRNA targeting Neomycin. On day 6, cells were cotransfected with a mixture of three plasmids: one expressing the F-Luc-5BoxB mRNA, another expressing Renilla luciferase (R-Luc), and a third expressing the indicated λN-HA-tagged proteins. (A) Northern blot analysis of representative RNA samples. (B) Western blot analysis of control and NOT1-depleted cells. α-Tubulin served as a loading control. Dilutions of control cell lysates were loaded in lanes 1–4 to estimate the efficacy of the depletion. (C) Firefly luciferase activity was normalized to that of Renilla luciferase. For each condition, the normalized values of F-Luc activity were set to 100 in cells expressing the λN-HA tag. Mean values ± standard deviations from three independent experiments are shown. (D) The normalized F-luc activity values were divided by the corresponding normalized mRNA levels for each condition. These ratios were set to 100 in cells expressing λN-HA.