3.1 Chemicals
As far as not otherwise stated, chemicals were purchased from Sigma-Aldrich (St. Louis, USA), Carl Roth (Karlsruhe, Germany), Merck (Darmstadt, Germany), Alfa Aesar (Haverhill, USA) or Thermo Fisher Scientific (Waltham, USA). Substances were either of analytical grade or had a purity ≥95 %. Organic solvents (HPLC/LC-MS grade) used for MS experiments were bought from VWR (Radnor, USA), Merck, Honeywell (Charlotte, USA), AppliChem (Darmstadt, Germany) or Biosolve (Valkenswaard, Netherlands).
3.2 Consumables
Consumables were bought from Sarstedt (Nümbrecht, Germany), Corning (Falcon®) (Corning, USA), Greiner Bio-One (Kremsmünster, Austria) or Beckman Coulter (Brea, USA) as far as not otherwise mentioned. For mass spectrometry experiments, plastic materials were purchased from Corning (Axygen®), Sarstedt or STARLAB (Hamburg, Germany).
3.3 Enzymes
Enzyme Manufacturer
Arylsulfatase A Rentschler Biopharma (Laupheim, Germany)
BamHI (Fast Digest) Thermo Fisher Scientific (Waltham, USA)
Calf Intestine Alkaline Phosphatase Invitrogen (Carlsbad, USA)
DpnI Thermo Fisher Scientific (Waltham, USA)
LysArginase Structural Biology Unit CSIC (Barcelona, Spain)
LysN Thermo Fisher Scientific (Waltham, USA)
Pfu DNA Polymerase Thermo Fisher Scientific (Waltham, USA)
Phusion DNA Polymerase Thermo Fisher Scientific (Waltham, USA)
Proteinase K Carl Roth (Karlsruhe, Germany)
RnaseA Thermo Fisher Scientific (Waltham, USA)
T4-Polynucleotide Kinase Thermo Fisher Scientific (Waltham, USA)
Trypsin (MS-Experiments) Promega (Fitchburg, USA)
Trypsin-EDTA solution (cell culture) life technologiesTM (Carlsbad, USA)
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3.4 Antibodies
3.4.1 Primary antibodies
Antigen Host Dilution Application Manufacturer
Cathepsin D rabbit 1:1000 WB Abcam (Cambridge, UK); Order
Number: ab97499
CREB rabbit 1:1,000 WB Cell Signaling Technology (Danvers,
USA); Order Number: 9197S human ASA (Antiserum) rabbit 1:2000 ELISA self-made by AG Matzner human MPR300 Dom9
N1+N2 del*
rabbit 1:1000 WB self-made by Tilman Schuster
LAMP-2 mouse 1:50 ICC Hybridoma Bank (Iowa City, USA);
Order Number: H4B4
p-CREB rabbit 1:1,000 WB Cell Signaling Technology (Danvers,
USA) ; Order Number: 9198S
SNAPIN rabbit 1:500 WB Proteintrech (Manchester, UK); Order
Number: 10055-1-AP
α-Tubulin mouse
1:10,000-1:20,000
WB Sigma-Aldrich (St. Louis, USA); Order Number: T5168
* Dom9 N1+N2 Del: domain 9 with deletion of two glycosylation sites
3.4.2 Secondary antibodies
Antigen Tag Host Dilution Application Manufacturer
mouse IgG (H+L) HRP goat 1:5,000 WB Jackson ImmunoResearch
(Pennsylvania, USA);
Order Number: 115-035-044 rabbit IgG (H+L) HRP goat 1:5,000 WB Jackson ImmunoResearch
(Pennsylvania, USA);
Order Number: 111-035-003 rabbit IgG (H+L) HRP goat 1:2,000 ELISA Jackson ImmunoResearch
(Pennsylvania, USA);
Order Number: 111-035-003
mouse IgG (H+L) Cy3 goat 1:250 ICC Jackson ImmunoResearch
(Pennsylvania, USA);
Order Number: 115-165-003
3.5 Cells
Bacterial Strain Genotype
E. coli XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F ́ proAB lacIqZ∆M15 Tn10 (Tetr)]
Mammalian Cell Lines Supplier
HEK 293 DSMZ-German Collection of Microorganisms and Cell Culture (Braunschweig, Germany)
HeLa DSMZ-German Collection of Microorganisms and Cell Culture (Braunschweig, Germany)
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3.6 Peptides
Sequence Supplier
FSEEKHLEDLER Dr. D. Winter (IBMB)
MHTGHANAKHIAGLSR JPT, Berlin, Germany
3.7 Primers
All primers were synthesized by Biomers (Ulm, Germany).
Name Sequence Application
SNAPIN S133A fwd
GGAATTTACCCCCCTGGCGCTCCAGGCAAAAGCGGACCG SDM SNAPIN
S133A SNAPIN
S133A rev
CGGTCCGCTTTTGCCTGGAGCGCCAGGGGGGTAAATTCC SDM SNAPIN
S133A SNAPIN
S133E fwd
GGAATTTACCCCCCTGGCGAGCCAGGCAAAAGCGGACCG SDM SNAPIN
S133E SNAPIN
S133E rev
CGGTCCGCTTTTGCCTGGCTCGCCAGGGGGGTAAATTCC SDM SNAPIN
S133E
pCMV fwd CCGTTGACGCAAATGGG Sequencing
pCMV-vector SNAPIN fwd
CRISPR-Cas9 1st PCR
ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTTGGCAAAAACCTAAGGTCCTG 1st PCR gene KO valid.
SNAPIN rev CRISPR-Cas9 1st PCR
TGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAACTGATTCGAGGCGGGGAGGA 1st PCR gene KO valid.
D501_long Primer A
AATGATACGGCGACCACCGAGATCTACACTATAGCCTACACTCTTTCCCTACACGACGCT 2nd PCR gene KO valid.
D502_long Primer A
AATGATACGGCGACCACCGAGATCTACACATAGAGGCACACTCTTTCCCTACACGACGCT 2nd PCR gene KO valid.
D503_long Primer A
AATGATACGGCGACCACCGAGATCTACACCCTATCCTACACTCTTTCCCTACACGACGCT 2nd PCR gene KO valid.
D701_long Primer B
CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTGGAGTTCAGACGTGTGCT 2nd PCR gene KO valid.
D702_long Primer B
CAAGCAGAAGACGGCATACGAGATTCTCCGGAGTGACTGGAGTTCAGACGTGTGCT 2nd PCR gene KO valid.
D703_long Primer B
CAAGCAGAAGACGGCATACGAGATAATGAGCGGTGACTGGAGTTCAGACGTGTGCT 2nd PCR gene KO valid.
D704_long Primer B
CAAGCAGAAGACGGCATACGAGATGGAATCTCGTGACTGGAGTTCAGACGTGTGCT 2nd PCR gene KO valid.
D705_long Primer B
CAAGCAGAAGACGGCATACGAGATTTCTGAATGTGACTGGAGTTCAGACGTGTGCT 2nd PCR gene KO valid.
D706_long Primer B
CAAGCAGAAGACGGCATACGAGATACGAATTCGTGACTGGAGTTCAGACGTGTGCT 2nd PCR gene KO valid.
D707_long Primer B
CAAGCAGAAGACGGCATACGAGATAGCTTCAGGTGACTGGAGTTCAGACGTGTGCT 2nd PCR gene KO valid.
D708_long Primer B
CAAGCAGAAGACGGCATACGAGATGCGCATTAGTGACTGGAGTTCAGACGTGTGCT 2nd PCR gene KO valid.
D709_long Primer B
CAAGCAGAAGACGGCATACGAGATCATAGCCGGTGACTGGAGTTCAGACGTGTGCT 2nd PCR gene KO valid.
D710_long Primer B
CAAGCAGAAGACGGCATACGAGATTTCGCGGAGTGACTGGAGTTCAGACGTGTGCT 2nd PCR gene KO valid.
D711_long Primer B
CAAGCAGAAGACGGCATACGAGATGCGCGAGAGTGACTGGAGTTCAGACGTGTGCT 2nd PCR gene KO valid.
D712_long Primer B
CAAGCAGAAGACGGCATACGAGATCTATCGCTGTGACTGGAGTTCAGACGTGTGCT 2nd PCR gene KO valid.
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3.8 Vectors
Name Construct Supplier
pCMV6_SNAPIN SNAPIN Myc-DDK Origene (Rockville, USA)
pCMV6_SNAPIN_S133A SNAPIN S133A Myc-DDK self-made SDM (Template: pCMV6_SNAPIN) pCMV6_SNAPIN_S133E SNAPIN S133E Myc-DDK self-made SDM (Template: pCMV6_SNAPIN) Edit-R Cas9 nuclease
expression plasmid (PuroR)
Cas9 Dharmacon/Horizon (Cambridge, UK),
3.9 Instruments
Instrument Model Manufacturer
Centrifuge (max. 2 ml) Z 216 MK Hermle(Wehingen, Germany)
Centrifuge benchtop Allegra X-15R Beckman Coulter (Brea, USA) Centrifuge cell culture Labofuge 400e Heraeus (Hanau, Germany) Counting Camber Fuchs-Rosenthal Brand (Wertheim, Germany) Cryopreservation system MVE TS; Tank 800
series-190
CHART (Luxembourg, Luxembourg) Detection system agarose gels BioDoc Analyze Biometra/Analytik Jena (Jena, Germany) Detection system HRP-based
immune detection
Fusion Solo Vilbert Lourmat (Collégien, France) Dounce homogenizer 7 ml, small clearance pestle Kimble Chase (Rockwood, USA) Electrotransfer system Trans-Blot® Cell Bio-Rad (Hercules, USA) Electrotransfer system PerfectBlueTM Semi-Dry
Electro Blotter
peqlab (Erlangen, Germany) Electrotransfer system Trans-Blot® Turbo Bio-Rad (Hercules, USA) Equipment agarose gel
electrophoresis
PerfectBlueTM Gel System peqlab (Erlangen, Germany) Equipment isoelectic focusing PROTEAN® IEF cell Bio-Rad (Hercules, USA) Equipment SDS-PAGE
electrophoresis (self-made gels)
Mini-PROTEON® Tetra Cell Bio-Rad (Hercules, USA)
Equipment SDS-PAGE electrophoresis (precast gels)
Mini Gel Tank life technologiesTM (Carlsbad, USA) Fluorescence microscope LSM 980 Airyscan 2
detector, inverse AxioObserver 7
Zeiss (Oberkochen, Germany)
Freeze dryer Lyovac GT2 Finn-Aqua (Hürth, Germany)
Heating block MHR23, BM15 Ditabis (Pforzheim, Germany)
Ice machine Ziegra (Isernhagen, Germany)
Incubator bacteria Function Line B12 Heraeus (Hanau, Germany)
Incubator bacteria (shaking) InnovaTM 43 New Brunswick Scientific (Edison, USA) Incubator cell culture HERAcell® 240 Heraeus (Hanau, Germany)
Laboratory rocker Rotamax 120 Heidolph (Schwabach, Germany)
Laminar flow cabinet Clean-Air Bio-Flow (Meckenheim, Germany) Liquid chromatography system Ultimate 3000 RSLC nano
HPLC system
Dionex (Idstein, Germany) Magnetic stand lysosome
enrichment
Quadro MACS Separator Miltenyi Biotec (Bergisch Gladbach, Germany) Magnetic stirrer WiseStir® MSH-20D Witeg (Wertheim, Germany)
Mass spectrometer LTQ Orbitrap Velos Thermo Fisher Scientific (Bremen, Germany) Mass spectrometer Orbitrap Fusion Lumos Thermo Fisher Scientific (Bremen, Germany) Mass spectrometer MALDI-TOF/TOF Autoflex III Bruker Daltonik (Bremen, Germany)
Micro scale CP1245-DCE Sartorius (Göttingen, Germany)
Micropipettes Gilson (Middleton, USA)
Microplate reader GENios Tecan (Männedorf, Switzerland)
Microplate reader Infinite Pro 2000 Tecan (Männedorf, Switzerland) Microplate washer HydroFlexTM Tecan (Männedorf, Switzerland) Microscope cell culture Axio Vert.A1 Zeiss (Oberkochen, Germany)
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Instrument Model Manufacturer
Multi-channel micropipette Transferpette S-12 Brand (Wertheim, Germany)
Orbital shaker PTR-35 Grant-bio (Cambridge, UK)
pH electrode WTW SenTix 50 Xylem (Weilheim, Germany)
pH meter pH-Meter 761 calimatic Knick (Berlin, Germany)
Power supply PowerPac 300 Bio-Rad (Hercules, USA)
Spectrophotometer NanoDrop 2000 Thermo Fisher Scientific (Waltham, USA) Thermal cycler T3 Thermalcycler Biometra/Analytik Jena (Jena, Germany)
Tip sonicator Sonifier 250 Branson (Danbury, USA)
Tip sonicator UP50H Hielscher (Teltow, Germany)
Tube Rotator LD79 Labinco (Breda, Netherlands)
Ultracentrifuge Optima TLX Beckman Coulter (Brea, USA)
Ultrasonic bath 2510 Branson (Danbury, USA)
Vacuum centrifuge ScanSpeed 40; Rotor GRV-m2.0-200
ScanVac/Labogene (Lynge, Denmark)
Vortex ZX3 UniEquip (Planegg, Germany)
Water bath cell culture 1083 GFL (Burgwedel, Germany)
Water purification system arium® Pro Sartorius, Göttingen, Germany
3.10 Software
Software Application Supplier
Capt Advance Solo 4 Software Fusion Solo HRP-based immune detection system; Signal
quantification
Vilbert Lourmat (Collégien, France)
Chromeleon Controlling Dionex
LC-system
Thermo Fisher Scientific(Waltham, USA) CorelDRAW Home & Student 2019 Corel Corporation (Ottawa, Canada)
Excel 2016 Data analysis and
visualization
Microsoft (Redmond, USA) flexAnalysis MALDI TOF data analysis Bruker Daltonik (Bremen, Germany) flexControl MALDI TOF data acquisition Bruker Daltonik (Bremen, Germany) Genome Compiler Primer design and Sanger
sequencing analysis
Genome Compiler Corporation (Los Altos, USA);
www.genomecompiler.com i-controlTM Software Infinite Pro 200
Microplate Reader
Tecan (Männedorf, Switzerland)
ImageJ Maximum intensity
projection LSM Z-stacks
Schneider et al. 2012
MaxQuant Orbitrap data analysis Cox and Mann 2008; Cox et al. 2011
NetPhos Kinase consensus sequence
analysis
Blom et al. 1999; Blom et al. 2004;
www.cbs.dtu.dk/services/NetPhos
OutKnocker NGS data analysis Schmid-Burgk et al. 2014; www.outknocker.org PEAKs Orbitrap data analysis Bioinformatics Solutions Inc. (Waterloo, Canada) Proteome Discoverer Orbitrap data analysis Thermo Fisher Scientific (Waltham, USA)
R-studio Data analysis and
visualization; statistics
R Core Team 2019 UCSC Genome Browser Determination of SNAPIN
reference sequence (NGS data analysis)
Kent et al. 2002; www.genome.ucsc.edu
Word 2016 Text processing program Microsoft (Redmond, USA)
Xcalibur Orbitrap data I Thermo Fisher Scientific (Waltham, USA)
Xfluor4 Software GENios
Microplate Reader
Tecan (Männedorf, Switzerland)
Zen Image Acquisition LSM 980
Airyscan 2
Zeiss (Oberkochen, Germany)
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