Tub gal4

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Tub-Gal4 is a genetic tool used in research. It functions as a transcriptional activator, driving the expression of target genes in specific cell types or tissues.

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8 protocols using tub gal4

Drosophila Genetics and Imaging Protocol

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All flies used in experiments were reared and maintained at 25°C on cornmeal fly food with dark/light cycle. For most experiments, white pre-pupae (0 h APF) of the desired genotype were collected and staged at 25°C. Following fly lines that are described in FlyBase and were obtained from the Bloomington Drosophila stock center (BDSC), the Vienna Drosophila Resource Center (VDRC), or the Kyoto Drosophila Genetic and Genomic Resources Center (DGGR), unless noted otherwise were used: UAS-EB1-GFP (BDSC 35512), Ecad-mTomato (BDSC 58789), UAS-Tub-EOS (BDSC 51313), Arm-GFP (BDSC 8555), tubGal4 (BDSC 5138), UAS-Lifeact-GFP (BDSC 35544), FRT42DtubGal80 (II), FRT42Dsspey⁰⁵²⁵² (DGRC 114436), hsFLP, Tub-Gal4 (BDSC 5138), UAS-GFP (BDSC 5430) sqh-EYFP-Golgi (BDSC 7193), His2avD-mRFP (BDSC 23651), Patronin RNAi (VDRC108927), Ubi-beta-TubGFP (DGRC 109604), UAS-palmKate2-K7 (attP2) (III) from Stefan Luschnig (University of Münster, Germany), GFP-Patronin22H2-N (attP40) (III) (Takeda et al., 2018 (link)), ciGal4 (Croker et al., 2006 (link)), and Ubi-p63E-cnn-RFP (Conduit et al., 2010 (link)).
For clonal analysis patronin^ey05252 FRT42D mutant positively marked clones were generated using FLP-mediated recombination. Clones were induced by heat-shock of prepua (0 h APF) at 37°C for 10 min in a water bath.

Panzade S, & Matis M. (2021). The Microtubule Minus-End Binding Protein Patronin Is Required for the Epithelial Remodeling in the Drosophila Abdomen. Frontiers in Cell and Developmental Biology, 9, 682083.

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Drosophila Genetic Stock Database

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All the genotypes analysed in this study are listed in Supplementary Table 1, in supplementary information. The following stocks were obtained from the Bloomington Drosophila Stock Center (BDSC), the Vienna Drosophila Resource Center (VDRC) or are described in Flybase: ci-GAL4 (Flybase #FBti0076751), hh-GAL4 (Flybase #FBti0017278), rn GAL4 (BDSC #7405), tub-GAL4 (BDSC #5138), pdm2-GAL4 (BDSC #49828), nub-GAL4 (Flybase #FBti0150342), FRT42D (BDSC #1802), FRT42D ubi-GFP (BDSC #5626), TRE-GFP (BDSC #59010), UAS-GFP (BDSC #35786), UAS-FLP (BDSC #4540), UAS-P35 (Flybase #FBtp0001646), UAS-HTT25Q Cerulean (BDSC #58360), UAS-HTT96Q-Cerulean (BDSC #56772) UAS-GFP-mCherry-Atg8a (BDSC #37749), UAS-Rheb (BDSC #9688), UAS-GADD34 (BDSC #76250), UAS-Atg1^RNAi (BDSC #44034), UAS-Rheb^RNAⁱ (BDSC #33966), UAS-Xrp1^RNAi (BDSC #34521), UAS Rpn2^RNAi (VDRC #106457), UAS-Rpt6^RNA (VDRC #49244), RPL5^2d2 (BDSC #25907), RPL14¹ (BDSC #2247), RPS13¹ (BDSC #2246), Dp(2:3)Cam14T (BDSC #4519).

Recasens-Alvarez C., Alexandre C., Kirkpatrick J., Nojima H., Huels D.J., Snijders A.P, & Vincent J.P. (2021). Ribosomopathy-associated mutations cause proteotoxic stress that is alleviated by TOR inhibition. Nature cell biology, 23(2), 127-135.

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Generating Transgenic Drosophila Strains

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Flies were grown on standard fly medium supplemented with yeast. NSlmb-3G86.32 was cloned in pUAS_attB to generate transgenic flies using the zh-86Fb landing platform (Bischof et al., 2007 (link)). Beside this strain, the following fly strains were used in this study: en-Gal4 (Bloomington Drosophila Stock Center (BDSC)), tub-Gal4 (BDSC), UAS_mCherry-NLS (Caussinus et al., 2008 (link)), ubiHis2Av::EYFP (Rebollo and González, 2004 (link)), sqhAX3; sqhSqh::GFP (Royou et al., 2004 (link)) and Gal4^332.3 (BDSC).

Brauchle M., Hansen S., Caussinus E., Lenard A., Ochoa-Espinosa A., Scholz O., Sprecher S.G., Plückthun A, & Affolter M. (2014). Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry. Biology Open, 3(12), 1252-1261.

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Drosophila Genetic Stock Database

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Drosophila Culture and Starvation Conditions

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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).

Liu Y., Mattila J, & Hietakangas V. (2020). Systematic Screen for Drosophila Transcriptional Regulators Phosphorylated in Response to Insulin/mTOR Pathway. G3: Genes|Genomes|Genetics, 10(8), 2843-2849.

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Investigating the Role of dRECS1 in Drosophila

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All experiments were performed with D. melanogaster grown at 25°C unless otherwise indicated. We generated transgenic animals allowing the Gal4/UAS (72 (link)) guided expression of the full-length (dRECS1) or truncated version (dRECS1^ΔC) of the CG9722/dRECS1 gene or the human RECS1 coding sequence (hRECS1), all of them tagged with a 3xFlag sequence at the N-terminal region. All additional Drosophila strains were obtained from Bloomington Drosophila Stock Center: w¹¹¹⁸ (BDSC:3605), y¹,w* (BDSC:1495), CG9722-KO [y¹,w*; Mi{MIC}CG9722^MI08483, BDSC:44979], UAS-CG9722-IR (TRiP.JF02887, BDSC:28052), UAS-CD8-GFP (BDSC:5130), UAS-GFP-mCherry-Atg8a (BDSC:37749), Cg-Gal4 (BDSC:7011), nub-Gal4 (BDSC:86108), and Tub-Gal4 (BDSC:5138).

Pihán P., Lisbona F., Borgonovo J., Edwards-Jorquera S., Nunes-Hasler P., Castillo K., Kepp O., Urra H., Saarnio S., Vihinen H., Carreras-Sureda A., Forveille S., Sauvat A., De Giorgis D., Pupo A., Rodríguez D.A., Quarato G., Sagredo A., Lourido F., Letai A., Latorre R., Kroemer G., Demaurex N., Jokitalo E., Concha M.L., Glavic Á., Green D.R, & Hetz C. (2021). Control of lysosomal-mediated cell death by the pH-dependent calcium channel RECS1. Science Advances, 7(46), eabe5469.

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Investigating Drosophila DNA Damage Repair

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The following fly stocks were used in this study: UAS-DDB1, UAS-DDB1^RNAi-Res, ddb1^HK-2-3, ddb1^W197 (generated in this study), ddb1^5-1, cul4^G1-3, cul4^JJ11, UAS-Flag-Cul4, UAS-Flag-Cul4^KR (CT Chien); mahj¹ [36 (link)]; UAS-Myc-Mahj, UAS-Myc-Mahj^R1120/1123E (this study). The following fly strains were obtained from BDSC: β-gal^RNAi (#50680), UAS-CD8-GFP (#32186), wts^RNAi (#34064), mahj^RNAi1 (#34912) and UAS-Yki^S168A (#28818); RNAi lines including ddb1^RNAi (#44974) and cul4^RNAi1 (#105668), cul4^RNAi2 (#44829) and mahj^RNAi2 (#110669) were obtained from the VDRC. Df(2R) XE2900 (#108418) is from the Kyoto Drosophila Genomics and Genetic Resource.
NSC drivers included insc-Gal4 (BDSC#8751; 1407-Gal4) or insc-Gal4, tub-Gal80^ts. Glial driver was repo-Gal4 (BDSC# 7145). Ubiquitous driver was tub-Gal4 (BDSC#5138). UAS-Dcr2 (BDSC#24650) was used together with various RNAi stocks. ddb1 and mahj RNAi knockdown efficiency was verified by immunostaining of anti-DDB1 and anti-Mahj antibodies in larval brains.
All experiments with mutants were carried out at 25 °C, and experiments for RNAi knockdown or overexpression were performed at 29 °C.

Ly P.T., Tan Y.S., Koe C.T., Zhang Y., Xie G., Endow S., Deng W.M., Yu F, & Wang H. (2019). CRL4Mahj E3 ubiquitin ligase promotes neural stem cell reactivation. PLoS Biology, 17(6), e3000276.

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Drosophila Maintenance and Genetic Stocks

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Flies were maintained at 25 C on medium containing malt 6.5% (w/v), semolina 3.2% (w/v), dry baker's yeast 1.8% (w/v), agar 0.6% (w/v), propionic acid 0.7% (v/v) and Nipagin (methylparaben) 2.4% (v/v) (Havula et al., 2013) or were grown on modified food containing 0.5% (w/v) agar, 2.5% (v/v) Nipagin (methylparaben) in PBS and supplemented with 20% (w/v) dry baker's yeast or the indicated concentrations of sucrose. Stocks used included UAS-Rheb (BDSC, 9688), UAS-InR (BDSC, 8262), UAS-Myc (Johnston et al., 1999) , UAS-Myc-RNAi (VDRC, 2947) TOR delP (BDSC, 7014), tub-Gal4 (Lee et al., 1999) , en-Gal4 (Fietz et al., 1995) , cg-Gal4 (BDSC, 7011), UAS-dPWP1-RNAi (NIG-Fly, 6751R-1 and 6751R-3), nclb 1 and nclb 2 (Casper et al., 2011) , HsFLP;;FRT82 GFP/ TM3, W;;Actin

Liu Y., Mattila J., Ventelä S., Yadav L., Zhang W., Lamichane N., Sundström J., Kauko O., Grénman R., Varjosalo M., Westermarck J, & Hietakangas V. (2017). PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression. Developmental cell, 43(2).

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