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Fig. 3. Expression of Cyclin E in the germarium. (A,B) Spectrin (red) marks the spectrosome in the germline stem cell and the fusome in the developing cyst. (A) Cyclin E protein expression (green) in the wild-type ovary is observed in region 1 of the germarium and sharply decreases in regions 2A and 2B. Asynchronous expression of Cyclin E resumes in region 3 and persists in the vitellarium. In wild-type germaria, Cyclin E expression oscillates strongly (inset): some cysts show Cyclin E expression (arrow), while other cysts are depleted of Cyclin E protein (arrowhead). (B) In encore mutant germaria, Cyclin E expression is observed throughout the germarium. There is no downregulation in region 2A/B. In encore mutant germaria, all cysts express some Cyclin E protein all the time (inset). (C) Reducing cyclin E gene dose in an encore mutant background suppresses the encore 32-cell egg chamber phenotype from 70 to 10% in females raised at 29°C. (D-F) Western blots of germaria-enriched extracts. (D) The accumulation of Cyclin A observed in encore mutant females can be partly restored to more normal levels by reducing cyclin E gene dose. The blot was stained with the protein dye ponceau (pink) as loading control. (E) The inhibition of proteolysis (Pase Inh.) or/and 19S-RP isopeptidase activity (NEM/Llnl) reveals that in encore mutant extracts Cyclin E accumulates as a high molecular form protein (arrowheads). The blot was stained with the protein dye ponceau (pink) as loading control. (F) Immunoprecipitation assays using anti-Drosophila Cyclin E antibodies followed by IB using anti-Ubiquitin antibodies shows more Cyclin E-Ubn in the encore mutant extract lane compared with wild type. The reciprocal immunoprecipitation assay shows more Cyclin E-Ubn in the encore mutant lane. Extract concentration of the starting material is shown stained with Coloidal blue Coomassie. The control lane (co) represents immunoprecipitation using wild-type extracts and an unrelated antibody raised in the same animal as the immunoprecipitation test.