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Fig. 5. Differential effect of TCE mutations on translational repression during oogenesis and embryogenesis. (A) Northern analysis of total RNA from nos-tub3'UTR (tub), nos-tub:TCE (TCE) or nos-tub:TCE mutant (TCEIIA, TCE[SRE^–], TCEIIIA) ovaries. Transgene RNAs, detected with a nos probe, were normalized to the rp49 control to determine their relative abundance, indicated below. (B) Immunoblot analysis of HA-Nos protein in extracts of stage 14 egg chambers from wild-type (WT), nos-tub3'UTR (tub), nos-tub:TCE (TCE) or nos-tub:TCE mutant derivatives (TCEIIA, TCE[SRE^–], TCEIIIA) using an anti-HA antibody. The antibody crossreacts with a protein that co-migrates with HA-Nos, as well as a more rapidly migrating protein in all samples. Snf protein was monitored as a loading control. In stage 14 oocytes, the wild-type TCE and stem-loop II mutants (TCEIIA, TCE[SRE^–]) prevent accumulation of HA-Nos, whereas stem-loop III mutants (TCEIIIA and others shown in Fig. S2) do not. Analysis of total ovarian extract [TCE(total)] confirms that nos-tub:TCE RNA is expressed and translated at earlier stages of oogenesis. (C) Immunoblot analysis of HA-Nos protein in extracts of stage 10 (st10) egg chambers and stage 14 (st14) oocytes dissected from nos-tub:TCE (TCE) or wild-type ovaries. (D) Immunoblot analysis of HA-Nos protein in extracts of total ovary or stage 14 (st14) oocytes from nos-tub:TCE females mutant for smg [smg¹/Df(Scf^R6)]. (E) Immunoblot analysis of HA-Nos protein in extracts of embryos from the same transgenic and wild-type lines shown in B. HA-Nos protein is detected in early embryos for all of the TCE mutants.

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