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Fig. 7. Ttv and Sotv form a complex and are co-localized in vivo. (A-C) Co-immunoprecipitation of Ttv with Sotv (A), of Ttv with Botv (B), and of Sotv with Botv (C). Drosophila S2 cells were transfected with plasmids to express Myc-tagged Ttv, V5-tagged Sotv and HA-tagged Botv in various combinations. Cell lysates were immunoprecipitated and then analyzed by western blotting with the antibodies indicated. IP, immunoprecipitation; IB, immunoblot. The `m' in the first lane of the left panel (A) indicates that ttv-myc and sotv-V5 were individually transfected and the cell lysates mixed in vitro prior to immunoprecipitation. Ttv-Myc and Sotv-V5 can easily precipitate each other (A), whereas Botv-HA cannot precipitate Ttv-myc or Sotv-V5, or vice versa (B,C). (D-D'') Double staining of Ttv-Myc (green) and a Golgi marker (red) in Drosophila S2 cells expressing Ttv-Myc. Ttv-Myc is concentrated in the Golgi complex. (E,E') Double staining of Ttv-Myc (green) and the ER marker Calnexin (red) in Drosophila S2 cells expressing Ttv-Myc. Ttv-Myc is co-localized with Calnexin. (F-F''') Co-staining of Ttv-myc (green), Sotv-V5 (blue) and Botv-HA (red) in S2 cells transfected with all three expression constructs. Although the majority of all three proteins are co-localized, Ttv-myc and Sotv-V5 appear to be more precisely co-localized and concentrated in certain compartments, whereas Botv-HA seems more uniformly distributed. (G-J) All embryos are oriented anterior left, dorsal up. All panels show embryos derived from females with corresponding mutant germline clones. (G) A ttv⁶³ null embryo. (H-J) Ectopic expression of ttv-myc driven by hairy^Gal4 (IJ3) in null embryos of ttv⁶³ (H), sotv⁴⁴ (I) and botv (J). X-gal staining was used to identify sotv (I) or botv (J) null embryos expressing UAS-ttv-myc by hairy^Gal4 (I,J). Embryos stained blue were selected for cuticle preparation. The genotypes shown are ttv⁶³/ttv⁶³UAS-nlacZ; IJ3 UAS-ttv-myc/+ (H), sotv⁴⁴/sotv⁴⁴ UAS-nlacZ; IJ3 UAS-ttv-myc/+ (I) and botv¹⁰³/botv¹⁰³ UAS-nlacZ; IJ3 UAS-ttv-myc/+ (J). Although ectopic expression of ttv-myc can rescue ttv⁶³ null with full penetrance, it cannot rescue sotv⁴⁴ and botv¹⁰³ mutant embryos.

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