Uas gfp rnai

Flies were reared on standard food at room temperature, 18°C, 25°C, or 31°C as indicated. Overexpression and transgenic RNAi studies were performed using the UAS/GAL4 system (RNAi crosses grown at 25°C or 31°C). Nephrocyte indicates the subtype of garland cell nephrocytes throughout the manuscript. Stocks obtained from the Bloomington Drosophila Stock Center (BDSC) were UAS-nephrin(sns)-RNAi (#64872), UAS-Rab5-RNAi (#34832), UAS-Rab5^S43N (dominant negative) (#42704), UAS-YFP-Rab5^Q88L (constitutively active, #9774), UAS-Rab7-RNAi (#27051), UAS-YFP-Rab7^T22N (dominant negative #9778), UAS-Rab11-RNAi (#42709), UAS-flo2-RNAi (#40833), and Shibire^ts (#2248). The second UAS-flo2-RNAi (#330316) and UAS-nephrin(sns)-RNAi-2 (VDRC #109442) were provided by the Vienna Drosophila RNAi Center (VDRC), prospero-GAL4 (Weavers et al., 2009 (link)) and Dorothy-GAL4 (#6903; BDSC) were used with or without tub-GAL80^ts (#7018; BDSC) to control expression in nephrocytes. UAS-GFP-RNAi (#41553; BDSC) or wild-type (yw¹¹¹⁸) were crossed to GAL4-drivers as control.

The following Drosophila melanogaster lines were used in this study: Oregon-R (Bloomington Drosophila Stock Center (BDSC) #6362), da-Gal4 (BDSC#55851), UAS-GFP RNAi (BDSC#9330), UAS-Ada2b RNAi (National Institute of Genetics, Japan (NIG) #9638R-3), white¹¹¹⁸ (w) mutant (BDSC#3605), hmlΔ-Gal4 UAS-2XEGFP (BDSC#30140), and Ada2b^d272 mutant (a gift from Dr. N. Zsindely, University of Szeged) [24 (link),25 (link)] lines. To knock down Ada2b ubiquitously, the da-Gal4 line was crossed with the UAS-Ada2b RNAi line. The resulting progenies (da-Gal4, UAS-Ada2b RNAi; designated as da > Ada2b RNAi) were used for the experiment. The progenies resulting from the cross of da-Gal4 and UAS-GFP RNAi lines were used as controls (da-Gal4, UAS-GFP RNAi; designated as da > GFP RNAi). To knock down Ada2b in hemocytes, the hmlΔ-Gal4 line was used in a similar way. The Ada2b mutant heterozygotes (Ada2b^d272/+) were obtained by crossing the w line with the w; Ada2b^d272/TM6 line. The siblings (TM6/+) of this cross were used as controls. Flies were reared on standard corn meal medium at 25°C.