Tub gal4

The following strains of D. melanogaster were used: w¹¹¹⁸; tub-GAL4, driver strain from Bloomington Drosophila Stock Center (y¹, w¹¹¹⁸; P{w^+mC = tubP-GAL4}LL7 P{ry^+t7.2 = neoFRT}82B/TM6B, Tb¹); P{KK109908}VIE-260B, KK RNAi strain from the Vienna Drosophila Resource Center, carrying a transgenic construct for expression of dsRNA for the Gagr gene RNA interference under UAS region control. Fly stocks were maintained in a standard nutrient agar medium at 25 °C. To induce interference, females of the UAS-Gagr RNAi strain were crossed with males of the tub-GAL4 driver strain. Thus, analyzed hybrids with knockdown of the Gagr gene, which we called Gagr^RNAi, were obtained. Females of the w¹¹¹⁸ strain were crossed with tub-GAL4 males as a control.

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)].

The following fly lines were used: w¹¹¹⁸ (background control), yw (background control), FB-Gal4 (Schmid et al., 2014 (link)), tub-Gal4 (Bloomington Stock Center no. 5138), yolk-Gal4 (Bloomington Stock Center no. 58814), UAS-LipinRNAi (VDRC transformant ID 36007), UAS-rel (Bloomington Stock Center no.9459), UAS-Toll^10b (Bloomington Stock Center no. 58987), plin1³⁸ (Bi et al., 2012 (link)), UAS-plin1 (Bi et al., 2012 (link)), UAS-TK RNAi (Bloomington Stock Center no. 25800), UAS-wRNAi (Bloomington Stock Center no. 28980), TKg-Gal4 (Song et al., 2014 (link)), PGRP-LE¹¹² (Bloomington Stock Center no. 33055), PGRP-LC^ΔE (Bloomington Stock Center no. 55713). Genetic recombination was used to generate UAS-plin1; tub-Gal4.

Targeted insertion of gce transgenes into the attP2 landing site on the third chromosome (cytological position 68A4) was achieved using the bacteriophage ϕC31 integrase method [44 (link)]. The pUASTattB constructs containing the WT and mutated gce or Met sequences were injected into embryos of the y w P{nos-ϕC31\int.NLS}X; P{CaryP}attP2 host strain (Genetic Services, Inc. or BestGene, Inc.). Several independent transgenic lines for expression of the FLAG-tagged Gce^WT, Gce^T272Y, and Gce^V315F proteins were generated through conventional P-element mediated transformation by injecting embryos of the w¹¹¹⁸ host strain with the pTFW-based vectors (Genetic Services, Inc.). In all cases, expression of the transgenic proteins was induced using the Gal4/UAS system [51 (link)] with the ubiquitous armadillo (arm-Gal4) or α-tubulin (tub-Gal4) drivers (Bloomington Drosophila Stock Center, Indiana).

All Drosophila melanogaster stocks, and crosses were maintained using standard medium at 25°C. The fly strains were, srp³ (BL2485), srp^AS (BL59020), Tub-Gal4 (BL5138), w¹¹¹⁸ (used as wild type background, BL3605), attP2 (BL25710) from the Bloomington Drosophila Stock Center, UAS-dsUsh (GD5712) (from Vienna Drosophila Resource Center) and srp^6G (Reuter, 1994 (link)). The strains ush^VX22 and ush^Rev24 were supplied by P. Heitzler. The fly lines msn-mCherry and Tj-Gal4 were kindly provided by the R. Schulz lab and Luisa Di Stefano, respectively.