Table 1.
Reagent resources and list of primers
| Reagent or Resource | Source | Identifier |
|---|---|---|
| Antibodies | ||
| 4E-BP antibody sampler kit | Cell Signaling | Cat# 9955T; RRID: AB_823409 |
| Rabbit monoclonal anti-Akt (11E7) (Pan) | Cell Signaling | Cat#4685S; RRID: AB_2225340 |
| Rabbit monoclonal anti-phospho-Akt (S473) (D9E) | Cell Signaling | Cat#4060; RRID: AB_2315049 |
| Rabbit monoclonal anti-phospho-Akt (Thr308) (D25E6) | Cell Signaling | Cat#13038; RRID: AB_2629477 |
| Rabbit polyclonal anti-Atg13 | Proteintech | Cat#18258-1-AP; RRID: AB_2130658 |
| Rabbit monoclonal anti-phospho-Atg13 (Ser355) (E4D3T) | Cell Signaling | Cat#46329; RRID: AB_ |
| Rabbit polyclonal anti-Atg14/Barkor (N-treminal) | Proteintech | Cat#19491-1-AP; RRID: AB_10642701 |
| Rabbit monoclonal anti-phospho-Atg14 (Ser29) (D4B8M) | Cell Signaling | Cat#92340; RRID: AB_2800182 |
| Mouse monoclonal anti-CD68 (3F7D3) | Abcam | Cat# ab201973; RRID: AB_ |
| Rat monoclonal anti-Ctip2 | Abcam | Cat# ab18465; RRID: AB_2064130 |
| Rabbit polyclonal anti-CUX1 | Proteintech | Cat#11733-1-AP; RRID: AB_2086995 |
| Mouse monoclonal anti-GFAP (GA5) | Cell signaling | Cat#3670; RRID: AB_561049 |
| Mouse monoclonal anti-GSK-3ß (3D10) | Cell signaling | Cat#9832; RRID: AB_ |
| Rabbit monoclonal anti-GSK-3ß (D5C5Z) | Cell signaling | Cat#12456; RRID: AB_2636978 |
| Rabbit monoclonal anti-phospho-GSK-3ß (Ser9) (D85E12) | Cell signaling | Cat#5558; RRID: AB_ |
| Rabbit polyclonal anti-IBA1 | Invitrogen | Cat# PA5-27436; RRID: AB_ |
| Rabbit monoclonal IGF-1 receptor ß (D23H3) | Cell signaling | Cat#9750; RRID: |
| Mouse monoclonal anti-LAMP2 | Abcam | Cat# ab25631; RRID: AB_470709 |
| Rabbit monoclonal anti-LAMTOR1/C11 orf59 (D11H6) | Cell signaling | Cat#8975; RRID: AB_10860252 |
| Rabbit monoclonal anti-LAMTOR4/C7 orf59 (D4P6O) | Cell signaling | Cat#13140; RRID: AB_2798129 |
| Rabbit monoclonal Anti-LAMTOR5/HBXIP (D4V4S) | Cell signaling | Cat#14633; RRID: AB_2798547 |
| Rabbit monoclonal anti-LC3A/B (D3U4C) | Cell signaling | Cat#12741; RRID: AB_2617131 |
| Mouse monoclonal anti-Myc-Tag (9B11) | Cell signaling | Cat#2276; RRID: AB_331783 |
| Rabbit polyclonal anti-Na,K-ATPase | Cell signaling | Cat#3010; RRID: AB_2060983 |
| Mouse monoclonal anti-NeuN (E4M5P) | Cell signaling | Cat#94403; RRID: AB_2904530 |
| Rabbit monoclonal anti-PI3 Kinase p110α (C73F8) | Cell signaling | Cat#4249; RRID: AB_2165248 |
| Rabbit polyclonal anti-SQSTM1/p62 | Abcam | Cat#ab91526; RRID: AB_2050336 |
| Phospho-TSC2 antibody sampler kit | Cell signaling | Cat#8350; RRID: AB_10949106 |
| Rabbit polyclonal anti-phospho-PI3 Kinase p85 (Tyr458) p55 (Tyr199) | Cell signaling | Cat#4228; RRID: AB_659940 |
| Rabbit monoclonal anti-PRAS40 (D23C7) | Cell signaling | Cat#2691; RRID: AB_2225033 |
| Rabbit monoclonal anti-phospho-PRAS40 (Thr246) (C77D7) | Cell signaling | Cat#2997; RRID: AB_2258110 |
| Rabbit monoclonal anti-PTEN (D4.3) | Cell signaling | Cat#9188; RRID: AB_2253290 |
| Rabbit monoclonal anti-phospho-PTEN (Ser380/Thr382/383) (44A7) | Cell signaling | Cat#9549; RRID: AB_659891 |
| Rag and Lamtor antibody sampler kit | Cell signaling | Cat#8665; RRID: AB_ |
| Rabbit monoclonal anti-Rheb (E1G1R) | Cell signaling | Cat#13879; RRID: AB_2721022 |
| Rabbit monoclonal anti-p70 S6 kinase (49D7) | Cell signaling | Cat#2708; RRID: AB_390722 |
| Rabbit polyclonal anti-phospho- p70 S6 kinase (Thr389) | Cell signaling | Cat#9205; RRID: AB_330944 |
| Mouse monoclonal anti-β-Actin (8H10D10) | Cell signaling | Cat#3700; RRID: AB_2242334 |
| Rabbit monoclonal anti-TBC1D7 (D8K1Y) | Cell signaling | Cat#14949; RRID: AB_2749838 |
| Rabbit monoclonal anti-Hamartin/TSC1 (D43E2) | Cell signaling | Cat#6935; RRID: AB_10860420 |
| Rabbit monoclonal anti-ULK1 (D8H5) | Cell signaling | Cat#8054; RRID: AB_11178688 |
| Rabbit monoclonal anti-phospho-UKL1 (Ser757) (D7O6U) | Cell signaling | Cat#14202; RRID: AB_2665508 |
| Chemicals | ||
| B27 supplement | Invitrogen | Cat# 17504044 |
| GlutaMAX | Invitrogen | Cat#35050 |
| N-[6-(biotinamido) hexyl]-3′-(2′-pyridyldithio) propionamide-biotin | Thermo Fisher Scientific | Cat#21341 |
| PBS | Thermo Fisher Scientific | Cat#10010023 |
| Benzonase | Sigma-Aldrich | Cat# E1014 |
| β-Mercaptoethanol | Sigma-Aldrich | Cat #M6250 |
| Na2CO3 | Sigma-Aldrich | Cat#222321 |
| NaHCO3 | Sigma-Aldrich | Cat#1063290 |
| Hepes | Quality Biologicals | Cat#118–089–721 |
| EDTA | Corning | Cat#46–034-CI |
| Paraformaldehyde | Electron Microscopy Sciences | Cat#15710 |
| PEG-400 | Med Chem Express | Cat# HY-Y0873A |
| Afuresertib | Med Chem Express | Cat# HY-15727 |
| Uprosertib | Med Chem Express | Cat# HY-15965 |
| A-674563 | Selleckchem | Cat#S2670 |
| MK2206 | Selleckchem | Cat#S1078 |
| Recombinant Human PPT1 | Creative Biomart | Cat#PPT1-367H |
| RPMI | Gibco | Cat#11875-093 |
| Fetal bovine serum | Hyclone | Cat#SH30071.03 |
| Pen-Strep | Gibco | Cat#15140-122 |
| Fatty acid–free BSA | ChemCruz | Cat#SC-2323A |
| Halt protease inhibitor | Thermo Fisher Scientific | Cat#1861279 |
| Halt protease-phosphatase inhibitor | Thermo Fisher Scientific | Cat#-78440 |
| LDS sample buffer | Thermo Fisher Scientific | Cat# NP0007 |
| RIPA buffer | Thermo Fisher Scientific | Cat#89901 |
| N-Ethylmaleimide | Sigma-Aldrich | Cat#E3876 |
| Hydroxylamine hydrochloride | Sigma-Aldrich | Cat#159417 |
| Thiopropyl Sepharose | GE-Amersham | Cat#17-0420-01 |
| SuperSignal West Pico PLUS chemiluminescent substrate | Pierce, Thermo Fisher Scientific | Cat#34578 |
| SuperSignal West Femto maximum sensitivity substrate | Pierce, Thermo Fisher Scientific | Cat#34096 |
| Methanol | Sigma-Aldrich | Cat#34860 |
| Triton X-100 | Sigma-Aldrich | Cat#T8787 |
| TrueBlack® lipofuscin autofluorescence quencher | Biotium | Cat # 23007 |
| DAPI-fluoromount G | Thermo Fisher Scientific | Cat #010020 |
| Cresyl violet stain solution | Abcam | Cat#ab246817 |
| Sucrose | Sigma-Aldrich | Cat#S0389 |
| Critical commercial kits | ||
| Neuronal dissociation kit | Miltenyl Biotec | Cat#130-094-802 |
| Neuron isolation kit | Miltenyl Biotec | Cat#130-115-389 |
| Plasma membrane isolation kit | Abcam | Cat# ab65400 |
| Lysosome isolation kit | Sigma=-Aldrich | Cat#LYSISO1 |
| RNA easy Mini kit | QIAGEN | |
| Superscript III First–strand synthesis kit | Invitrogen | Cat#18080051 |
| RNeasy Mini Kit | Qiagen | Cat#74104 |
| IGF1 ELISA kits | Thermo Fisher Scientific | Cat# EMIGF1 |
| RheB activation assay kit | New East Biosciences | Cat# 81201 |
| Insulin ELISA kits | Thermo Fisher Scientific | Cat# EMINS |
| H&E kit | Abcam | Cat#ab245880 |
| Primers used for Lamtor1 mutagenesis | ||
| Lamtor1-C3A-5 CTTGAAGGAATTCGGTACCATGGGGGCCTGCTATAGCAGCGAAAACGAGGACTC |
||
| Lamtor1-C3A-3 GAGTCCTCGTTTTCGCTGCTATAGCAGGCCCCCATGGTACCGAATTCCTTCAAG |
||
| Lamtor1-C4A-5 GAAGGAATTCGGTACCATGGGGTGCGCCTATAGCAGCGAAAACGAGGACTCGG Lamtor1-C4A-3 CCGAGTCCTCGTTTTCGCTGCTATAGGCGCACCCCATGGTACCGAATTCCTTC |
||
| C3AC4A-5 GGCTTGAAGGAATTCGGTACCATGGGGGCCGCCTATAGCAGCGAAAACGAGGACTCGGAC C3AC4A-3 GTCCGAGTCCTCGTTTTCGCTGCTATAGGCGGCCCCCATGGTACCGAATTCCTTCAAGCC |
||
| Primers used for qRT-PCR | ||
| Lamtor1 F-CATTGATGTGTCTGCCGCAGAC R-CTTCCAATGGGTCAGACTGCTG |
||
| Lamtor2 F-ACCAAGCGTTTAATGAAGACAGTC R-TGAGCATTCCGAAGCCTACGGT |
||
| Lamtor3 F-AGACCTGGCTTCCTATCCACGT R-AACTCACCACCAGAGGCAAACG |
||
| Lamtor4 F-AGTGCCATCTCGGAGTTGGTCA R-ACCTCGGTTCTGCCTCTTCACT |
||
| Lamtor5 F-GTCCTATGCACAGATTCACAAGG R-ACATACCACAGGGATGTCGGTG |
| Primers used for qRT-PCR | Source | Identifier |
|---|---|---|
| Rag A F-TGCAAAGAGCAGCGAGATGT R-CATGCTCTGGAAAGAAGCGG |
||
| Rag B F-ATCGCGTCCGCTAGAATGTT R-TGGGCATCTCGCTGTTCTTT |
||
| Rag C F-GCAGTCATCAGACCAGTGCT R-CAGCAGAGAAACTCCACCGT |
||
| Rag D F-CCTTTGCAGTCACCTTGTGC R-GTGTTTTAGACCCCTCGGCA |
||
| GAPDH F-TGGCCTCCAAGGAGTAAGAA R-TGTGAGGGAGATGCTCAGTG |
||
| Plasmid | ||
| Myc-tagged Lamtor1 | GeneCopoeia | Cat#EX-Mm19894-M09 |
| Software and algorithm | ||
| ABI Prism software version 1.01 | Applied Biosystems | N/A |
| Image quant IQTL software | GE Healthcare Lifesciences | N/A |
| ZenDesk | Zeiss | N/A |
BSA, bovine serum albumin; DAPI, 4',6-diamidino--2-phenylindole; IGF1, insulin-like growth factor-1; PPT1, palmitoyl-protein thioesterase-1; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; RIPA, radioimmunoprecipitation assay.