Uas rheb

The lines of jhamt^RNAi (103237 and 19172), Fpps^RNAi (104362), Sni^RNAi (106219 and 27342), Cyp305a1^RNAi (101644 and 51486), Met^RNAi (100638 and 10801), gce^RNAi (11176), tai^RNAi (15709), Jhe^RNAi (44049) and PTEN^RNAi (109278) were obtained from VDRC. Met^RNAi (61935), gce^RNAi (combination of 61852 and 26323 at the same time), tai^RNAi (36095), UAS-PI3K (8287), UAS-Rheb (9689), UAS-S6KII (8714), 10xSTAT92E-GFP (26197), UAS-EGFP-NiPp1 (23712) and Aug21Gal4 (30137) were obtained from Bloomington Stock Center. UAS Diap-1 was a gift from G. Morata. UAS-AstC was generated cloning the EcoRI-KpnI fragment from the cDNA clone RH36507 (DGC gold BDGP) into the pUAST vector. Transgene expression together with UAS-GFP was driven by esg-Gal4, Dl-Gal4, Su(H)-Gal4 or pros-Gal4. Gal4 activity was regulated by Tub > Gal80^ts. Flies were crossed at 17 °C, and two day old progeny was transferred to 29 °C for analysis. MARCM clones were generated by a 1hr heat shock at 37 °C of 2–5 days old females and were marked by the tubulin > Gal4 line driving the expression of UAS GFP (normal clones). Apc-Ras clones were generated as described previously^{15 (link)}.

For most experiments Drosophila strains were cultured in standard cornmeal-agar medium (#789211, NutriFly BF, Genesee Scientific) and maintained at 25 °C. The RNAi experiments were performed at 28 °C as previously described [35 (link)]. The sau^z2217 mutant strain was described previously [35 (link), 67 (link)]. UAS-dGOLPH3 RNAi (#46150, [35 (link)]) and UAS-Tctp RNAi (# 26632) were obtained from the Vienna Drosophila Resource Center Collection (VDRC, [44 (link)]). The following fly strains were from the Bloomington Drosophila Stock Center (BDSC, Indiana University, Bloomington, IN, USA): UAS-Rheb (#9689), UAS-GFP (#4775), Df(2 L)Exel7010 (#7782), en-Gal4 (#30564), nub-Gal4 (#86108), ey-Gal4 (#5534), elav-Gal4 (#8765), and bam-Gal4 (#80579, [68 (link)]). The following fly strains were obtained from FlyORF (University of Zurich, [69 (link)]): UAS-dGOLPH3-HA (#F002769), UAS-Tctp-HA (#F002479), UAS-14-3-3ζ-HA (#F001064). bam-Gal4 and tub-Gal4/tub-Gal80^ts (gift of Dr. Timothy Megraw, Florida State University, USA) were used to deplete dGOLPH3 respectively in spermatocytes and in larval/pupal tissues and to express dGOLPH3-HA, Tctp-HA, and 14-3-3ζ-HA proteins. The fly strains expressing either GFP, or fluorescent tagged-dGOLPH3 were described previously: GFP [34 ], dGOLPH3-RFP [34 ], GFP-dGOLPH3 [35 (link)], and GFP-dGOLPH3^K167A/R170L [35 (link)].

Fly stocks were maintained at room temperature (approximately 22 °C) in yeast extract/cornmeal/molasses/agar food medium. w¹¹¹⁸ was used as wild type control to cross with esg^ts > GFP in different experiments. Transgenic RNAi fly stocks used were: bun RNAi1 (VDRC19679), bun RNAi2 (VDRC19680), Madm RNAi1 (VDRC27346), Madm RNAi2 (VDRC27347), hpo (VDRC104169, TRiP27661), msn (TRiP28791), TSC2 (VDRC6313). Transgenic fly stocks UAS-InR^A1325D, UAS-EGFR^A887T, UAS-Notch^DN, UAS-Vein, UAS-Upd3 and UAS-Upd has been previously described [8 (link), 9 (link), 11 (link), 17 (link)]. UAS-Rheb is from Bloomington (9689). The Madm fly stocks FRT82BMadm^3T4, FRT82BMadm^2D2 were previously described [36 (link)]. The bun and Madm fly stocks UAS-BunA, UAS-BunB, UAS-Madm, UAS-Madm;UAS-BunA, FRT40AΔGE12921, FRT40Abun^200B, FRT40ΔGE12921;UAS-Ras^V12, FRT40Abun^200B;UAS-Ras^V12 were previously described [27 (link), 28 (link), 36 (link)]. UAS-Ras^v12 on III was a gift from Nam Moon.