Reagents and tools table
| Reagent/Resource | Reference or Source | Catalog Number |
|---|---|---|
| Experimental Models | ||
| HeLa-M WT cells | Frazier et al, 2016 | N/A |
| AP4B1 KO Hela cells | Frazier et al, 2016 | N/A |
| SPG47 patients Fibroblasts | Provided by Professor H. Houlden (UCL) | N/A |
| Age/gender matched control fibroblasts | Provided by Professor H. Houlden (UCL) | N/A |
| SPG47 iPSC-derived neurons | 2-year-old male diagnosed with AP4B1-associated SPG47 | N/A |
| Control iPSC-derived neurons | Fibroblasts from the clinically-unaffected sex-matched parent (heterozygous variant) | N/A |
| SH-SY5Y WT cells | Davies et al, 2018 | N/A |
| SH-SY5Y AP4B1 KO cells | Davies et al, 2018 | N/A |
| C57BL-6 WT mice | Jackson Laboratory: C57BL/6J-Ap4b1em5Lutzy/J | Strain #: 031349 |
| C57BL-6 ap4b1 KO mice | Jackson Laboratory: C57BL/6J-Ap4b1em5Lutzy/J | Strain #: 031349 |
| Cynomolgus monkey (WT) | Novaxia Pathology Laboratory | N/A |
| Plasmids/Vectors | ||
| pTRS-KS-CBh | Provided by Dr S. Gray (University Texas Southwestern) | N/A |
| pAAV/CBh-MCS | Provided by Prof M. Robinson (University of Cambridge) | N/A |
| pLXIN-AP4B1 | Provided by Prof M. Robinson (University of Cambridge) | N/A |
| pLenti/PGK-MCS-Vos | Provided by Prof K. de Vos (University of Sheffield) | N/A |
| pCIneo_V5-N | Provided by Prof K. de Vos (University of Sheffield) | N/A |
| pHelper | PlasmidFactory | N/A |
| pAAV2/9 | PlasmidFactory | N/A |
| Pseudotyped AAV9 | Lukashchuk et al, 2016 | N/A |
| pAAV/CBh-V5-hAP4B1 | Fabricated in house | N/A |
| LV/V5-empty | Fabricated in house | N/A |
| LV/V5-AP4B1 | Fabricated in house | N/A |
| AAV9/V5-empty | Vector builder | N/A |
| AAV9/CBh-hAP4B1 | Cornell | N/A |
| AAV9/SYN-hAP4B1 | Vector Biolabs | N/A |
| AAV9/CBh-mAP4B1 | Vector Biolabs | N/A |
| Antibodies IF | ||
| Rabbit-anti-ATG9A | Abcam | Cat# ab108338 |
| Sheep-anti-TGN46 | Bio-Rad | Cat# AHP500G |
| Anti-Golgin 97 | Abcam | Cat# 169287 |
| Mouse-anti-beta-Tubulin III | Synaptic Systems | Cat# 302304 |
| Mouse-anti-beta-Tubulin III | Sigma | Cat# T8660 |
| Mouse-anti-calbindin | Abcam | Cat# ab82812 |
| Anti-rabbit-Alexa Fluor 488 | Thermo Fisher | Cat# A-11073 |
| Anti-sheep Alexa Fluor 558 | Thermo Fisher | Cat# A-11016 |
| Anti-mouse Alexa Fluor 647 | Thermo Fisher | Cat# A-21235 |
| Hoechst dye | Sigma | Cat# 94403 |
| Western Blot | ||
| Pre-cast 4–20% mini-PROTEAN gels | Bio-Rad | Cat# 4561095 |
| 245 kDa protein ladder | Abcam | Cat# ab116028 |
| Anti-mouse AP4E1 | BD Biosciences | Cat# 612019 |
| Anti-rabbit GAPDH | CST | Cat# 14C10 |
| Anti-rabbit IgG (HRP) | Thermo Fisher | 31460 |
| Anti-mouse IgG (HRP) | Thermo Fisher | 31430 |
| Oligonucleotides and other sequence-based reagents | ||
| M18s forward primer | 5′-GTAACCCGTTGAACCCCAT-3′ | N/A |
| M18s reverse primer | 5′-CCATCCAATCGGTAGTAGCG-3′ | N/A |
| hAP4B1 forward primer | 5′-CTGGTGAACGATGAGAATGT-3′ | N/A |
| hAP4B1 reverse primer | 5′-GACCCAGCAACTCTGTTAAA-3′ | N/A |
| Chemicals, Enzymes and other reagents | ||
| RIPA buffer | 50 mM Tris-HCL pH 6.8; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 0.1% v/v SDS; 0.5% v/v deoxycholic acid; 1% Triton X-100 | N/A |
| 1x protease inhibitor cocktail | Sigma-Aldrich | Cat #539136 |
| RNeasy® lipid tissue mini kit | QIAGEN | Cat #74804 |
| Proteinase K | Thermo-fisher | EO0491 |
| QIAzol® lysis reagent | QIAGEN | 79306 |
| NEBcloner kit | New England Biolabs | N/A |
| QIAEX II® gel extraction kit | QIAGEN | 20021 |
| QuantiNova SYBR® Green qPCR kit | QIAGEN | 208052 |
| Brilliant III Ultra-Fast SYBR® Green QPCR kit | QIAGEN | 600883 |
| Mouse IFN-γ ELISpot BASIC kit | Mabtech | #3321-2A |
| Histology | ||
| Charged glass slides | Starfrost | #MBB-0302-55A |
| Haematoxylin | Cellpath | #RBA-4205-ooA |
| Eosin solution | Leica | #3801590E |
| Xylene | Fisher | #X/0200/17 |
| DPX mounting medium | Cellpath | SEA-1300-00A |