Fig. 3. Sply expression. (A) Sply expression is developmentally regulated. Sply mRNA was quantified by RNase protection, as described in the Materials and Methods. Relative expression was determined using ImageQuant software and standardized to the intensity of a ribosomal protein subunit (RpL32) transcript. (B) In situ hybridization of wild-type embryos shows that Sply has strong, transient expression in the syncytial blastoderm (stage 4) that declines to undetectable levels after cellularization. At stage 11-12, Sply mRNA reappears in the midgut/hindgut rudiments where the developing gut is undergoing extensive reorganization. This gut expression persists for the duration of embryogenesis. The absence of any detectable staining in Sply⁰⁵⁰⁹¹ mutants under identical conditions demonstrates probe specificity and reaffirms that there is no Sply expression in this line. (C) Expression of Sply in wild-type (Canton-S), homozygous Sply mutant (Sply⁰⁵⁰⁹¹) and homozygous Sply revertant (Sply^14a) lines. RNA was obtained from 0-to 24-hour-old embryos. RpL32 is again used as a loading control. (D) Degradation of endogenous LCBPs. Extracts of wild-type (square) and Sply mutant (diamond) adult flies were analyzed for the ability to degrade Drosophila endogenous phosphorylated long chain bases.