PI3K & Rbsn-5 in Drosophila Figure 8. Rbsn-5 is a PI(3)P target protein. (A) Rbsn-5 (red) was co-localized in the pupal wing discs, mainly with GFP-2xFYVE, Rab5 and Rab7 (arrowheads), but not with Rab11. The scale bars represent 20 mm. (B) Rbsn-5 (red) was clearly reduced in the dVps15-knockdown region (the left sides of the dotted lines). In the knockdown region, DE-Cadherin (green) accumulated intracellularly as shown in Figure 4. (C) Adult wings in which dsRNAs were expressed by dpp-Gal4 driver. Malformation was observed near the third vein where the dsRNAs for dVps15, dVps34 or Rbsn-5 were expressed. This malformation was synergistically enhanced by co-expression of dsRNAs for both dVps15 and dVps34 or both dVps15 and Rbsn-5, whereas no such enhancement was observed by co-expression of dsRNAs for both PI3K class I and Rbsn-5 or both PI3K class I and dVps34. (D) Horizontal (upper) and vertical (lower) sections of the pupal wings expressing dsRNAs for Rbsn-5 and/or dVps15. b-integrin (red) was not localized in the double knockdown region (indicated by double headed arrow in the left panel) whereas marginal defects were observed in the single knockdown (indicated by double headed arrows in the middle and right panels). The right sides of the panels represent the internal control where b- integrin was normally localized to the basal junctions. The scale bars represent 10 mm. doi:10.1371/journal.pone.0007306.g008 (CG9746) dsRNA, UAS-Rbsn-5 (CG8506) dsRNA; UAS-PI3K Class I (CG4141) dsRNA, UAS-GFP-Rab5 (a gift from K. Matuno), UAS-dVps34 dsRNA; UAS-dVps34 cDNA, UAS-Rbsn- 5 cDNA; UAS-Rbsn-5 dsRNA, UAS-dVps34 dsRNA; UAS- dVps15 dsRNA, UAS-dVps34 dsRNA; UAS-Rbsn-5 dsRNA, UAS-PI3K Class I dsRNA; UAS-Rbsn-5 dsRNA, UAS-PI3K Class I dsRNA; UAS-dVps34 dsRNA. UAS-GFP-2xFYVE; UAS- dVps15 dsRNA, UAS-GFP-2xFYVE; UAS-Rbsn-5 dsRNA, UAS- GFP LAMP1; UAS-dVps15 dsRNA, UAS-GFP Rab5; UAS- dVps15 dsRNA, and driver strains were sd-GAL4 and dpp- GAL4. Fly transformation followed standard methodology. Clone generation Rbsn-5 mutant clones were generated by crossing y w2/Y; P{neoFRT}40A rbn5 c241/Cyo flies to y w2 hs-FLP; P{w+; Ubi- GFP(S65T)nls}2L P{neoFRT}40A/CyO flies. Progeny were heat shocked for 2 h at 37.5uC at either the second or third larvae stage. Flies were kept at 25uC up to the white pupal stage and then at 26.5uC until dissection. Immunofluorescence microscopy Staged pupae wings and 3rd larva wing discs were dissected and fixed with 4.0% (w/v) paraformaldehyde (PFA)/PBS solutions for PLoS ONE | www.plosone.org 10 October 2009 | Volume 4 | Issue 10 | e7306