Tub gal80ts

All flies were reared on standard cornmeal-based fly food. Unless noted otherwise, during developmental and post-eclosion, flies were raised at 25 °C, ~50% humidity and a 12:12 hr light-dark cycle (1400 ± 200 lx white fluorescent light) in humidity and temperature controlled incubators.
The following stocks were used: Canton-S (BL 64349), tub-Gal80^ts (BL 7019), UAS-CD8:GFP (BL 5130), 20XUAS-IVS-mCD8:GFP (BL 32194), UAS-dMBD-R2-RNAi (BL 30481), and UAS-dMBD2/3-RNAi (BL 35347) were obtained from the Bloomington Stock Center (Bloomington, IN). Drosophila virilis (15010–1051.00) and D. yakuba (14021.0261.38) was received from the Drosophila Species Stock Center (San Diego, CA). Cha-Gal80 was a gift from T. Kitamoto and Jay Hirsh generously provided the Tdc2-Gal4 line.

Drosophila were grown in a medium composed of corn meal/agar/yeast, the culture temperature was maintained at 24 ± 1 °C, and the light/dark cycle was 12 h. The fly stocks w¹¹¹⁸ (#3605), Tub-Gal80^ts; TM2/TM6B (#7019), Tub-Gal4/TM3, Sb1, Ser1 (#5138) were obtained from the Bloomington Drosophila Stock Center (Bloomington, IN, USA). To generate CR11538-overexpressing flies, the full-length lncRNA-CR11538 was constructed into the pUAST-attB vector using primers listed in Supplementary Table S1. The constructed plasmid was injected into embryos of attp2# (25C6): y [1 (link)] M{vas-int.Dm}ZH-2A w[*]; P{CaryP}attP40. In order to minimize the effect of lncRNA-CR11538 on the development of Drosophila, the transgenic CR11538 flies were crossed with temperature sensitive Tub-Gal80^ts; Tub-Gal4 flies and cultured at 18 °C, and transiently overexpressed lncRNA-CR11538 once transferred to an incubator at 29 °C for 24 h for the following experiments.

tub-GAL80^TS, w¹¹¹⁸, UAS-CD8GFP, and UAS-CD8-PARP1-Venus were obtained from the Bloomington Stock Center and repo-GAL4 and UAS-tau^WT (0N4R) were kindly provided by Dr. Mel Feany. Double recombinant control (repo-GAL4,tub-GAL80^TS) and triple recombinant tau transgenic (repo-GAL4,tub-GAL80^TS,UAS-tau^WT) flies were balanced over TM6B,Tb. Crosses were performed at 18°C or 28°C in incubators with humidity control and 12-hour light:dark cycles. Flies were maintained on standard Drosophila Bloomington food (Nutri-Fly, Genesee Scientific).

The generation of the ^UASBirA and ^UAS(^bioUb)₆-BirA flies and their recombination with elav^GAL4 for the studying of ubiquitinated material in the embryo nervous system development had been previously described [21 (link)]. For the analysis of the ubiquitinated material in Drosophila adult brain ^UASBirA and the ^UAS(^bioUb)₆-BirA flies were independently recombined with the eye specific Glass Multimer Reporter-GAL4 driver (GMR^GAL4). Flies carrying the GMR^GAL4 (BL 1104), the pan-neuronal elav-GAL4 (elav^GAL4; BL 8765) or the heat shock-GAL4 (Hs^GAL4; BL 2077) drivers on the second chromosome and flies carrying the tubulin-GAL4 (Tub^GAL4; BL 5138) and GAL80 (Tub^GAL80ts; BL 7017) drivers on the third chromosome were provided by the Bloomington Stock Center (Bloomington, IN, USA). Flies with the glutamatergic neuron-specific GAL4 driver (OK371^GAL4) on the second chromosome were kindly provided by Cahir O’Kane. Ube3a gain of function and loss of function mutants were a gift from Janice Fischer [34 (link)]. Flies overexpressing the C-terminal half of Rpn10 (^UASRpn10-ΔNTH) were a gift from Zoltan Lipinszki [35 (link)].

Flies were kept in standard fly vials (containing polenta and yeast) in a 26°C incubator. The following fly lines were used: dpp^FO, dpp^FO-GFP, dpp-Gal4, and UAS-FLP (Matthew Gibson), UAS-NLS-mCherry (Caussinus et al., 2008 (link)), ptc-Gal4 (w*; P{GawB}ptc559.1), P{act5C(FRT.polyA)lacZ.nls1}3, ry506, tub-Gal80ts (Bloomington stock center).

Strain y¹w¹¹⁸ was used as control. The following stocks were used: tub-GAL80ts (7108), UAS-Dicer (24651) and UAS-perd RNAi JF01159 (31584) from Bloomington Drosophila Stock Center (http://flystocks.bio.indiana.edu/). Kettin-GFP (ZCL2144) from FlyTrap (http://flytrap.med.yale.edu/). UAS-perd RNAi (106680) and UAS-mys RNAi (29619) from Vienna Drosophila RNAi Center (http://stockcenter.vdrc.at). Zasp-GFP (110740) from Drosophila Genetic Resource Center (http://www.dgrc.kit.ac.jp/). Mef2-GAL4 (Ranganayakulu et al., 1996 (link)). UAS-mCD8-GFP (Lee and Luo, 1999 (link)). UAS-rhea-mCherry (Venken et al., 2011 (link)). UAS-kon-HA (Schnorrer et al., 2007 (link)). 1151-GAL4 was a gift from Lingadahalli S. Shashidhara (Centre for Celular and Molecular Biology, Hyderabad, India). For transient expression assays, the different UAS lines were crossed with tub-GAL80ts; Mef2-GAL4 at 18°C, and the UAS expression was induced switching to 29°C. The temperature shift was made when larvae were between the first and the second larval stage, unless otherwise specified.

Reagents and fly strains used were as follows: w¹¹¹⁸ (#3605), ppl-Gal4 (#58768) and tubGal80^ts (#7017) were obtained from Bloomington Drosophila Stock Center (https://bdsc.indiana.edu), dilp2-Gal4 and UAS-dilp2 [41 (link)]. dilp2Gal4; tubGal80^ts [79 (link)] and pplGal4; tubGal80^ts lines were generated in the lab. UAS-bmm and UAS-lsd2 were provided by Dr. Ronald Kühnlein. All RNAi lines were procured from Vienna Drosophila RNAi Center (VDRC).