Fig. 2.

Prp8 primarily localizes to the cytoplasm in Drosophila cells. (A–D) Immunoblots showing comparable induction of Flag-tagged (A,B) and non-tagged (C,D) wild-type and RP-Prp8 variants in lysates prepared from the third instar larval EADs expressing the respective transgenes under the control of the GMR-Gal4 driver. The transgenic Prp8 proteins do not markedly elevate the total Prp8 levels (C,D). ATP5α served as loading controls. Data represent means±s.d. of normalized Prp8 protein expression, n=5 (B), n=3 (D). Statistical significance was determined using ordinary one-way ANOVA with Tukey's multiple comparisons test, n.s., non-significant. (E–P) Transfected Flag-tagged human PRPF8^wt (G), Drosophila Prp8^wt (F) and RP-Prp8 mutant proteins (H–N) showing cytoplasmic localization in Drosophila S2 cells (GFP) similar to the non-tagged Drosophila Prp8^wt (O) or endogenous Prp8 (P) as determined by immunostaining with an anti-Flag (E–N) or Prp8-specific antibodies (O,P). Expression of UAS-based Prp8 transgenes was driven by actin promoter from co-transfected pAW-Gal4 plasmid while GFP-expressing pIE-GFP vector served to identify transfected cells. Nuclei were stained with DAPI. Scale bars: 5 µm. (Q–R) Overexpressed non-tagged Prp8^wt (Q) and Prp8^S>F (R) transgenic proteins using dpp-Gal4 driver are enriched in cytoplasm of larval wing imaginal disc cells (Q‴, R‴). The dpp expression domain is marked by membrane-tethered RFP (Q,R); nuclei are stained with DAPI. Scale bars: 10 µm.