KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Mouse monoclonal anti-Parkin | Abcam | Cat# ab77924; RRID: AB_1566559 |
| Rabbit monoclonal anti-phospho-Ubiquitin | CST | Cat# 70973; RRID: AB_2799795 |
| Rabbit monoclonal anti-PINK1 | Abcam | Cat# ab216144; RRID: AB_2927726 |
| Rabbit monoclonal anti-pS6(pS235/236) | CST | Cat# 4856; RRID: AB_2181037 |
| Rabbit monoclonal anti-S6 | CST | Cat# 2217; RRID: AB_331355 |
| Rabbit monoclonal anti-GAPDH | CST | Cat# 5174; RRID: AB_10622025 |
| Mouse monoclonal anti-GAPDH | CST | Cat# 97166; RRID: AB_2756824 |
| Rabbit monoclonal anti-TOM20 Alexa Fluor 488 | Abcam | Cat# ab205486; RRID: AB_2943509 |
| Rabbit monoclonal anti-TOM20 Alexa Fluor 647 | Abcam | Cat# ab209606; RRID: AB_2934123 |
|
Biological samples | ||
| Healthy adult human brain tissue lysate (whole) | Novus Biologicals | Cat# NB820–59177 |
|
Chemicals, peptides, and recombinant proteins | ||
| Geneticin (G418 Sulfate) | Life Tech. Corp. | Cat# 10131035 |
| Doxycycline Hydrochloride | Sigma Aldrich | Cat# D3072 |
| SNAP-Cell 647-SiR | NEB | Cat# S9102 |
| Hoechst 33342 | Invitrogen | Cat# H2570 |
| carbonyl cyanide 3-chlorophenylhydrazone (CCCP) | Sigma Aldrich | Cat# C2759 |
| DMSO | Sigma Life Science | Cat# D2650 |
| Rapalog (A/C heterodimerizer; AP21967) | Takara Bio. | Cat# 635055 |
| CMPD39 (USP inhibitor 18) | MedChem Express | Cat# HY-141659 |
| Rapamycin | Sigma Aldrich | Cat# R0395 |
| Halt protease and phosphatase inhibitor cocktail | ThermoFisher | Cat# 78440 |
| Polybrene | EMD Millipore | Cat# TR-1003-G |
|
Critical commercial assays | ||
| Bio-Rad protein assay dye reagent concentrate (Bradford assay kit) | Bio-Rad | Cat# 5000006 |
| Mini-protean TGX 4–20% SDS-PAGE gels | Bio-Rad | Cat# 4561093 |
| Lipofectamine 3000 kit | Invitrogen | Cat# L3000–015 |
| NEBuilder HiFi DNA assembly master mix kit | NEB | Cat# E2621S |
| Q5 Site-directed mutagenesis kit | NEB | Cat# E0554S |
|
Deposited data | ||
| Plasmid maps, single-cell microscopy quantification data, and analysis code | This manuscript | Zenodo: https://doi.org/10.5281/zenodo.8356580 |
|
Experimental models: Cell lines | ||
| Human: HeLa cell line (female) | UCSF cell culture and banking core | Cat# CCLZR030; RRID: CVCL_0030 |
| Human: HEK293T cell line (female) | UCSF cell culture and banking core | Cat# CCLZR076; RRID: CVCL_0063 |
|
Oligonucleotides | ||
| mNeonGreen cDNA | Shaner et al.61 | Allele Biotech Cat# ABP-FP-MNEONSB |
|
Recombinant DNA | ||
| pCMV-Parkin | This manuscript | N/A |
| pCMV-Parkin(E16A) | This manuscript | N/A |
| pCMV-mKO2-Parkin(M1) | This manuscript | N/A |
| pCMV-mKO2-Parkin(M1L) | This manuscript | N/A |
| pCMV-Parkin-A92mKO2 | This manuscript | N/A |
| pCMV-Parkin(C431N)-A92mKO2 | This manuscript | N/A |
| pLv-EF1a-Parkin-A92mKO2 | This manuscript | N/A |
| pLv-EF1a-Parkin(S65A)-A92mKO2 | This manuscript | N/A |
| pLv-EF1a-Parkin(R104A)-A92mKO2 | This manuscript | N/A |
| pLv-EF1a-Parkin(K161N)-A92mKO2 | This manuscript | N/A |
| pLv-EF1a-Parkin(N273K)-A92mKO2 | This manuscript | N/A |
| pLv-EF1a-Parkin(W403A)-A92mKO2 | This manuscript | N/A |
| pLv-CMV(trunc)-Parkin-A92mKO2 | This manuscript | N/A |
| pLv-EF1a-SNAP-FRB-FIS1(MTS) | This manuscript | N/A |
| pLv-TetOn-FKBP-PINK1(110–581)-mNeonGreen (Neo) | This manuscript | N/A |
| pLv-EF1a-FKBP-PINK1(110–581)-mNeonGreen | This manuscript | N/A |
| pLv-EF1a-FKBP-PINK1(G386A) (110–581)-mNeonGreen | This manuscript | N/A |
| pLv-EF1a-FKBP-PINK1(S228A) (110–581)-mNeonGreen | This manuscript | N/A |
| pLv-EF1a-FKBP-PINK1(S228D) (110–581)-mNeonGreen | This manuscript | N/A |
| pCMV-mCherry-Parkin | Narendra et al.5 | Addgene Plasmid #23956 |
| pLL3.7m-Clover-Geminin(1–110)-IRES-mKO2-Cdt(30–120) | Bajar et al.62 | Addgene Plasmid #83841 |
| pLV-EF1a-IRES-Puro | Hayer et al.63 | Addgene Plasmid #85132 |
| pSNAPf | NEB | N9183S |
| pC4-RhE-FRB-Fis1 | Narendra et al.64 | Addgene Plasmid #68056 |
| pC4M-F2E-GFP-FKBP | Narendra et al.64 | Addgene Plasmid #68058 |
| pCMVTNT-PINK1-C-Myc | Beilina et al.65 | Addgene Plasmid #13314 |
| pLv-TetOn-MCS1-P2A-MCS2 (Neo) | Adam Karpf Lab | Addgene Plasmid #89180 |
| pLv-TetOn-FKBP-PINK1 (110–581)-mNG-Neo | This manuscript | N/A |
| pMD2.G | Didier Trono Lab | Addgene Plasmid #12259 |
| pCMV-dR8.91 | NovoPro | Cat# V007548 |
|
Software and algorithms | ||
| MATLAB | https://www.mathworks.com/ | N/A |
| Fiji | Schindelin et al.66 | https://imagej.net/software/fiji/ |
| filt2 MATLAB function | Greene67 | https://www.mathworks.com/matlabcentral/fileexchange/61003-filt2-2d-geospatial-data-filter |
| UCSF ChimeraX software | Pettersen et al.68 | https://www.cgl.ucsf.edu/chimerax/ |
| WebLogo: A sequence Logo generator | Crooks et al.69 | https://weblogo.berkeley.edu/logo.cgi |
| EMBL-EBI Clustal Omega multiple sequence alignment tool | Madeira et al.70 | https://www.ebi.ac.uk/Tools/msa/clustalo/ |