KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| LAMP1 Mouse monoclonal, H4A3 | Developmental Studies Hybridoma Bank | Cat# H4A3, RRID:AB_2296838 |
| mTOR (7C10) Rabbit mAb | Cell Signaling Technology | Cat# 2983, RRID:AB_2105622 |
| RagA (D8B5) Rabbit mAb | Cell Signaling Technology | Cat# 4357, RRID:AB_10545136 |
| RagC (D31G9) XP Rabbit mAb | Cell Signaling Technology | Cat# 5466, RRID:AB_10692651 |
| RHEB (E1G1R) Rabbit mAb | Cell Signaling Technology | Cat# 13879, RRID:AB_2721022 |
| TSC2 (D93F12) XP Rabbit mAb | Cell Signaling Technology | Cat# 4308, RRID:AB_10547134 |
| Phospho-Tuberin/TSC2 (Thr1462) Antibody | Cell Signaling Technology | Cat# 3611, RRID:AB_329855 |
| P70 S6 kinase (49D7) Rabbit mAb | Cell Signaling Technology | Cat# 2708, RRID:AB_390722 |
| Phospho-p70 S6K (T389) Rabbit mAb | Cell Signaling Technology | Cat# 9025, RRID:AB_2734746 |
| Phospho-Drosophila p70 S6K (T389) Rabbit Ab | Cell Signaling Technology | Cat# 9209, RRID:AB_2269804 |
| GAPDH (14C10) Rabbit mAb | Cell Signaling Technology | Cat# 2118, RRID:AB_561053 |
| β-Actin (D6A8) Rabbit mAb | Cell Signaling Technology | Cat# 8457, RRID:AB_10950489 |
| Mios (D12C6) Rabbit mAb | Cell Signaling Technology | Cat# 13557, RRID:AB_2798254 |
| WDR59 (D4Z7A) Rabbit mAb | Cell Signaling Technology | Cat# 53385, RRID:AB_2799432 |
| HA-Tag (C29F4) Rabbit mAb | Cell Signaling Technology | Cat# 3724, RRID:AB_1549585 |
| DYKDDDDK Tag (D6W5B) Rabbit mAb | Cell Signaling Technology | Cat# 14793, RRID:AB_2572291 |
| Myc-Tag (9B11) Mouse mAb | Cell Signaling Technology | Cat# 2276, RRID:AB_331783 |
| GST (26H1) Mouse mAb | Cell Signaling Technology | Cat# 2624, RRID:AB_2189875 |
| Anti-Rabbit IgG, HRP-linked | Cell Signaling Technology | Cat# 7074, RRID:AB_2099233 |
| Anti-Mouse IgG, HRP-linked | Cell Signaling Technology | Cat# 7076, RRID:AB_330924 |
| Akt (pan) (C67E7) Rabbit mAb | Cell Signaling Technology | Cat# 4691, RRID:AB_915783 |
| Phospho-Akt (Thr308) (D25E6) XP Rabbit mAb | Cell Signaling Technology | Cat# 13038, RRID:AB_2629447 |
| Phospho-Akt (Ser473) (D9E) XP Rabbit mAb | Cell Signaling Technology | Cat# 4060, RRID:AB_2315049 |
| Anti HaloTag Mouse mAb | Promega | Cat# G9211, RRID:AB_2688011 |
| Anti-Rabbit IgG AlexaFluor 488 conjugated | Thermo Fisher | Cat# A-11008, RRID:AB_143165 |
| Anti-Rabbit IgG AlexaFluor 594 conjugated | Thermo Fisher | Cat# A-11037, RRID:AB_2534095 |
| Anti-Mouse IgG AlexaFluor 488 conjugated | Thermo Fisher | Cat# A-11029, RRID:AB_138404 |
| Anti-Mouse IgG AlexaFluor 594 conjugated | Thermo Fisher | Cat# A-11005, RRID:AB_141372 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| Fibronectin human plasma | Sigma-Aldrich | Cat# F0895 |
| Paraformaldehyde | Electron Microscopy Sciences | Cat# 15714 |
| Triton X-100 | Sigma-Aldrich | Cat# T8787 |
| Disuccinimidyl suberate | Thermo Fisher | Cat# 21555 |
| Proteinases inhibitor cocktail | Thermo Fisher | Cat# 78430 |
| Phosphatases inhibitor cocktail | Thermo Fisher | Cat# 78420 |
| 4’,6-diamidino-2-phenylindole (DAPI) | Thermo Fisher | Cat# D1306 |
| Hoechst 33342 | Thermo Fisher | Cat# H3570 |
| Nocodazole | Sigma-Aldrich | Cat# M1404 |
| Critical Commercial Assays | ||
| Pierce™ c-Myc-Tag IP/Co-IP Kit | Thermo Fisher | Cat# 23630 |
| Halo-Trap kit | Chromotek | Cat# otak-20 |
| Experimental Models: Cell Lines | ||
| HeLa (Wild type) | ATCC | Cat# CCL-2 |
| HeLa, WDR24-KO | (Cai et al., 2016) | N/A |
| HeLa, WDR59-KO | This paper | N/A |
| HeLa, MIOS-KO | This paper | N/A |
| Experimental Models: Drosophila Stocks | ||
| seh1Δ15 | (Senger et al., 2011) | N/A |
| UAS-Wdr24 RNAi | Bloomington Drosophila Stock Center | Cat# 62393 |
| UAS-RagA RNAi | Bloomington Drosophila Stock Center | Cat# 34590 |
| UAS-RagC RNAi | Bloomington Drosophila Stock Center | Cat# 32342 |
| UAS-mCherry RNAi | Bloomington Drosophila Stock Center | Cat# 35787 |
| Nanos-Gal4 | (Van Doren et al., 1998) | N/A |
| TSC1-GFP | Vienna Drosophila Resource Center | Cat# 318377 |
| Lamp1–3xmCherry | (Hegedus et al., 2016) | N/A |
| Oligonucleotides | ||
| ON-TARGETplus Non-targeting siRNA control | GE Healthcare | D-001810-01-05 |
| SMARTpool: ON-TARGETplus LAMTOR1 siRNA | GE Healthcare | L-020916-02-0005 |
| SMARTpool: ON-TARGETplus RRAGA siRNA | GE Healthcare | L-016070-00-0005 |
| SMARTpool: ON-TARGETplus RRAGC siRNA | GE Healthcare | L-017822-01-0005 |
| Recombinant DNA | ||
| FLAG-TSC2 | (Tee et al., 2002) | Addgene plasmid #8996 |
| mPlum-LAMP1 | (Shaner et al., 2008) | Addgene plasmid #55982 |
| mEOS4b-TSC2 | This paper | N/A |
| Halo-TSC2 | This paper | N/A |
| Halo-TSC2 T1462A | This paper | N/A |
| Halo-TSC2 S939A | This paper | N/A |
| Halo-TSC2 T1462A/S939A | This paper | N/A |
| FLAG-RAGA(T21N) | (Shen et al., 2017) | Addgene plasmid #99715 |
| FLAG-RAGA(Q66L) | (Shen et al., 2017) | Addgene plasmid #99716 |
| FLAG-RAGC(Q120L) | (Shen et al., 2017) | Addgene plasmid #99725 |
| FLAG-RAGC(S75N) | (Shen et al., 2017) | Addgene plasmid #112757 |
| HA-GST-RHEB1 | (Sancak et al., 2007) | Addgene plasmid #14951 |
| Myc-mTOR | (Sarbassov et al., 2004) | Addgene plasmid #1861 |
| HA-RAGA | (Shen et al., 2017) | Addgene plasmid #99710 |
| FLAG-RAGC | (Shen et al., 2017) | Addgene plasmid #99723 |
| Software and Algorithms | ||
| Microsoft Excel | Microsoft | https://products.office.com/en-us/excel |
| GraphPad Prism 8 | GraphPad Software Inc. | https://www.graphpad.com/scientificsoftware/prism/ |
| Imaris 9.3.0 | Bitplane Inc. | https://imaris.oxinst.com/products/imaris-forcell-biologists |
| ImageJ | National Institutes of Health | https://imagej.nih.gov/ij/ |
| Photoshop CC | Adobe | https://www.adobe.com/products/photoshop.ht ml |
| Other | ||
| Dulbecco’s modified Eagle’s medium GlutaMAX™-I with pyruvate | Thermo Fisher | Cat# 10569010 |
| Fetal bovine serum | Thermo Fisher | Cat# A3160602 |
| Penicillin-Streptomycin | Thermo Fisher | Cat# 15140122 |
| DMEM without amino acids | MyBioSource | Cat# MBS653087 |
| Fetal bovine serum, dialyzed | Thermo Fisher | Cat# A3382001 |
| TrypLE™ Express | Thermo Fisher | Cat# 12605010 |
| Xfect™ Transfection reagent | Takara | Cat# 631318 |
| Xfect™ RNA Transfection reagent | Takara | Cat# 631450 |
| Dynabeads™ Protein A | Thermo Fisher | Cat# 1001D |
| M-PER mammalian protein extraction buffer | Thermo Fisher | Cat# 78503 |
| Pierce™ Protein-Free T20 (TBS) Blocking Buffer | Thermo Fisher | Cat# 37571 |
| Clarity™ Western ECL substrate | Biorad | Cat# 170–5060 |