Uas rheb

Flies were grown at 25°, on medium containing agar 0.6% (w/v), malt 6.5% (w/v), semolina 3.2% (w/v), baker’s yeast 1.8% (w/v), nipagin 2.4%, propionic acid 0.7%. In starvation experiments, larvae were kept on medium contain agar 0.5% (w/v), nipagin 2.5%, and propionic acid 0.7% in PBS, supplemented with or without 5% sucrose (w/v). Fly stocks used in this study were: Tub-Gal4 (Lee et al. 1999 (link)), Ey-Gal4 (BDSC 5534), w¹¹¹⁸ (BDSC 6326), NURF38 RNAi (BDSC 31341), UAS-Rheb (BDSC, 9688), mTOR delP mutant (BDSC 7014), myc dm² mutant (Maines et al. 2004 (link)), UAS-Myc (Johnston et al. 1999 (link)), UAS-Myc RNAi (VDRC 2947).

Flies were maintained at 25 ℃ and 50% humidity on a standard cornmeal food (the recipe for 1 L food is: 56 g cornmeal, 40 g yeast, 88 g glucose, 10 g agar, and 1.25 g nipagin) with a 12-h light/dark cycle. The Gal4/UAS (Upstream Activating Sequence) binary system [16 (link)] was applied to manipulate transgene expression in various tissues, with experimental crosses conducted at 25 ℃. FLP-out strains were crossed with indicated UAS-transgene strains at 25 ℃ to generate clones labeled with fluorescence. 5–7-day-old mated flies or 3rd instar larvae were used in all experiments, with detailed descriptions provided in the corresponding figure legends.
Fly strains utilized in this study: w¹¹¹⁸ (lab stock), lpp-Gal4 (lab stock), hsFLP; AyGal4, UAS-GFP (lab stock), hsFLP; AyGal4, UAS-RFP (lab stock), UAS-CG33474-3×Flag (this study), CG33474-Gal4 (this study), Ubi-GFP-PTS1 (this study), UAS-GFP (BDSC, #52262), UAS-RFP (BDSC, #8545), UAS-Luciferase-RNAi (BDSC, #31603), UAS-lacZ (lab stock), UAS-CG33474-RNAi (BDSC, #64672), UAS-Rheb-RNAi (BDSC, #33966), UAS-Pex19-RNAi (VDRC, #22064), UAS-bmm-RNAi (VDRC, #37877), UAS-Sod1-RNAi (BDSC, #34616), UAS-Sod2-RNAi (BDSC, #25969), UAS-Rheb (BDSC, #9688), and UAS-GFP-PTS1 (BDSC, #64248).

Fly stocks used in this study were: chro^8c (Wasser, M); chro^17a (Wasser, M); chro⁷¹ (Wasser, M); chro⁶¹² (Wasser, M); UAS-Chro (Wasser, M); pros¹⁷ (Doe, C.Q); UAS-Grh O (Bray SJ); grh³⁷⁰ (Bray SJ); pzg⁶⁶ (Nagel, A.C.); UAS-CycB (Lehner C.); UAS-CycE (Richardson H.); OK107-Gal4 (L. Luo); Jil-1^Z2 (K.M. Johansen). The following fly stocks were obtained from Bloomington Drosophila Stock Center (BDSC): UAS-InR.A1325D (InRCA: InR constitutively active, BDSC8440); UAS-Rheb (BDSC9689); UAS-PI3KCAAX (BDSC25908); chro deficiency (Df(3L)ED231; BDSC8090); grh deficiency (Df(2R)Pcl7B; BDSC3064); east RNAi (BDSC33879). chro RNAi (v101663) and pzg RNAi (v25542) were obtained from Vienna Drosophila Resource Center (VDRC). Mtor^k03905 was obtained from Kyoto Drosophila genetic resource center (DGRC 111163). To induce RNAi knockdown of various genotypes, Drosophila larvae were kept at 29 °C by insc-Gal4 unless otherwise stated. To generate transgenic fly stocks containing genomic DNA encompassing wild-type chro and its promoter region, a P(acman) BAC clone, CH322-159M1 was integrated into BDSC9736 (y¹ w¹¹¹⁸; PBac⁷VK00018) genome^{53 (link)}.
To deprive larvae of dietary amino acids, larvae hatching within a 2-h window were transferred into amino acids-free medium (5% sucrose, 1% agar in phosphate-buffered saline (PBS)) and raised at 29 °C for 3 days before larval brains were dissected.