Fig 1.

Nanos stimulates Pumilio-mediated repression. (A) Diagrams of Renilla (Rn) and firefly (FF) luciferase reporters with wild-type (NRE) or mutant (NREmut) Nanos response elements are depicted. (B) qRT-PCR analysis of Nanos (Nos), Pumilio (Pum), or positive-control Rpl32 mRNAs from D.mel-2 cells (cells only) or cells transfected with Nos expression plasmid. Average cycle threshold (Avg Ct) values are indicated below corresponding samples. NTC, no-template control reactions; N/D, not detected. (C) Percent repression by Nos, reported for the indicated mass of transfected plasmid, was calculated relative to mutant Nanos C354Y. See Fig. S2A in the supplemental material for raw data. (D) Percent repression of luciferase protein (dual-luciferase assay) and mRNA (qRT-PCR). See Table S1 in the supplemental material for the corresponding data. (E) Percent repression by Nanos in cells treated with dsRNA for LacZ (negative control), Nos, Pum, or (in panel F) Brain Tumor (Brat) and 4EHP. In each figure, Western blotting confirmed expression of V5 epitope-tagged proteins. Data for panels E and F are reported in Table S2 in the supplemental material. (G) Fluorescent imaging of SDS-polyacrylamide (4 to 20%)-separated TMR-labeled HaloTag (HT) Brat or 4EHP proteins. Protein depletion by dsRNA to Brat or 4EHP was calculated relative to control LacZ RNAi.