4.1 Antibodies
The antibodies used are listed in Table 1 with dilution in TBS‐T (10 mM Tris, pH7.6, 150 mM NaCl, 0.05% (v/v) Tween20) supplemented with 3% (w/v) BSA for primary antibodies or 5%
(w/v) milk powder for secondary and directly horseradish peroxidase (HRP) labelled antibodies, respectively.
species Type Company Dilution
‐Actin (Ac‐15) human mouse mAb Abcam 1:1000
c‐myc (Clone 9E10) human mouse mAb Sigma‐Aldrich 1:1000
FAT10 (4F1) human mouse mAb (Aichem, Pelzer
et al. 2010) 1:50000 GFP (Clones 7.1 and
13.1) human mouse mAb Roche 1:1000
GST (B 14: sc‐138) human mouse mAb Santa Cruz
Biotechnology 1:1000 GST‐FAT10 (105(3)) human rabbit pAb (Hipp, Raasi et al.
2004) 1:1000
Ubiquitin (FK2) human mouse mAb Santa Cruz
Biotechnology 1:2000
VCP ((5): sc‐57492) human mouse mAb Santa Cruz
Biotechnology 1:2000
Vinculin (hVIN‐1) human mouse mAb Sigma‐Aldrich 1:10000
mouse ab (AffiniPure
IgG) HRP mouse goat pAb Jackson
ImmunoResearch 1:5000 rabbit ab (AffiniPure
IgG) HRP rabbit goat pAb Jackson
ImmunoResearch 1:5000
6xHis (M2) HRP mouse mAb Sigma‐Aldrich 1:2000
HA (Clone HA‐7) HRP/
Agarose mouse mAb Sigma‐Aldrich 1:4000
Flag® (M2) HRP/
Agarose mouse mAb Sigma‐Aldrich 1:2000
4.2 Cell culture
Human embryonic kidney (HEK) 293 cells were cultivated in Iscove’s modified Dulbecco’s
medium (IMDM; Lonza) supplemented with 10% Fetal Calf Serum (FCS; Lonza), 5%
Ultraglutamine (Lonza), 100 U/mL Penicillin and 100 g/mL Streptomycin (Lonza) at 37 °C with 5% CO2.
HEK293 cells were seeded for experiments at 2.5 x 105 cells per 10 cm2 dish, 1x 106 cells per 60 cm2 dish and 2 x 106 cells per 145 cm2 dish and grown over night. For DNA transfection TransIT®‐LT1 transfection reagent (Mirus) in IMDM without supplements and for siRNA transfection X‐treme Gene® transfection reagent (Roche) in OptiMEM® (Life technology) was used. FAT10 expression was induced by addition of 200 U/mL IFN and 400 U/mL TNF
(Preprotech) to the medium. After 24 hours cells were treated with 10 M of the proteasome inhibitor MG132 (Enzo Lifesciences), 10 M of the VCP inhibitors Eeyarestatin I (EerI) or N2,N4‐dibenzylquinazoline‐2,4‐diamine (DBeQ) for 4 hours or 2.5 mg/mL Tunicamycin (Sigma) for 6 h before harvest. Cycloheximide chases were performed by treatment for 1, 2 and 4 hours with 50 g/mL cycloheximide.
4.3 Immunoprecipitation
24 h after DNA transfection or induction and 48 h after siRNA treatment cells were lysed in lysis buffer (20 mM Tris, pH 7.6, 50 mM NaCl, 10 mM MgCl2, 1% (v/v) NP‐40, 1 x Complete
mini® protease inhibitor (Roche)) for 30 min on ice. Lysates were centrifuged for 30 min at
20000 x g. 30 L of equilibrated Protein A sepharose beads (Sigma) and the respective antibody were added and incubated for 2 h at 4 °C on a roller. Beads were washed twice with NET‐TN buffer (50 mM Tris, pH 8.0, 650 mM NaCl, 5 mM EDTA, 0.5% (v/v) Triton X‐100) and twice with NET‐T buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% (v/v) Triton X‐
100), boiled in sample buffer (225 mM Tris, pH 6.8, 5% (w/v) SDS, 10% (v/v) ‐ME) and separated on NuPAGE® Bis‐Tris 4‐12% acrylamide gradient gels (Invitrogen) or 12.5%
acrylamide containing Läemmli gels. After sodium‐dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) proteins were transferred by western blotting to a nitrocellulose membrane (GE Healthcare) with the wet‐blot technique in Towbin‐buffer (25 mM Tris, pH8.3,
192 mM glycin, 20% (v/v) methanol) using 0.95 mA, 100 V and 300 W for 45 min. Membranes were incubated in Rotiblock (Roth) for 1 h at room temperature (RT) to block unspecific binding. Afterwards membranes were incubated with the respective primary antibodies for 1 h at RT, washed twice with TBS‐T (10 mM Tris, pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween 20) and incubated with the respective antibody for 1 h at RT. Directly labelled antibodies were incubated for 1.5 h at RT. Immunoblotting was vizualized using Clarity™ Western ECL Blotting Substrate (BioRad) in a ChemiDocTM system (BioRad).
4.4 Plasmids
For DNA transfection of HEK293 cells the plasmids listed in Table 2 were used.
Table 2: Plasmids for gene expression in cell lines.
Plasmid Source
pcDNA3.1‐HA‐FAT10 (Hipp, Raasi et al. 2004)
pcDNA3.1‐HA‐FAT10GG (Aichem, Pelzer et al. 2010)
pcDNA3.1‐His‐3xFlag‐FAT10 (Chiu, Sun et al. 2007)
pcDNA6‐FAT10 Nicola Catone, Kreuzlingen
pCMV‐myc‐VCP Dr. Annette Aichem, Kreuzlingen
pCMV‐myc‐VCP E578Q this work
pCMV‐HA‐EGFP this work
pEGFP‐N1‐HA‐FAT10 PD Dr. Gunter Schmidtke, Konstanz
pEGFP‐N1‐HA‐FAT10 N term PD Dr. Gunter Schmidtke, Konstanz
pEGFP‐N1‐HA‐FAT10 C term PD Dr. Gunter Schmidtke, Konstanz
pBi‐Tet‐HA‐Nub1L PD Dr. Gunter Schmidtke, Konstanz
pBi‐Tet‐TA Clontech
pcDNA3.1‐HA‐Nub1LUBA PD Dr. Gunter Schmidtke, Konstanz
pcDNA3.1‐HA‐Nub1LUBL PD Dr. Gunter Schmidtke, Konstanz
pcDNA3‐ss‐YFP2‐1‐Antitrypsin Prof. Dr. Hans‐Peter Hauri, Basel pcDNA3‐ss‐YFP2‐1‐Antitrypsin Z mutant Prof. Dr. Hans‐Peter Hauri, Basel
For production of recombinant proteins the constructs shown in Table 3 were transformed
site Gene Target
plasmid Aim
VCP pSumo Cloning of VCP for expression in bacteria
4.6 siRNA
The knockdown of endogenous FAT10 was achieved by transfection of the four siRNAs (40 nM each):
5’‐cccauaugacagcgugaaatt‐3’, 5’‐agcauucuuugucuauuaatt‐3’, 5’‐gggauuuaaugaccuuugatt‐3’, 5’‐gauugugacuugcaauggatt‐3’.
As unspecific control the AllStars Negative Control siRNA (40 nM; SI03650318, Qiagen) was used.
4.7 Quantitative real‐time PCR
For verification of the knockdown of endogenous FAT10 RNA from HEK293 cells was prepared using the RNeasy® Mini Kit (Qiagen) according to the manufacturer’s protocol. 1 g RNA was transcribed into cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). For the PCR reaction the TaqMan® Fast Universal Master Mix (Applied Biosystems) was used with FAM‐primers for FAT10 (Hs00197374_m1, life technologies) and the housekeeping gene GAPDH (Hs99999905_m1) in the 7900HT Fast Real‐time PCR system (Applied Biosystems) as recommended by the manufacturer.
4.8 Recombinant proteins
E.coli BL21(DE3) were transformed with the respective plasmids and grown in HSG medium (13.5 g/L peptone, 7 g/L yeast extract, 14.9 g/L glycerol, 2.5 g/L NaCl, 2.3 g/L K2HPO4, 1.5 g/L
KH2PO4, 0.14 g/L MgSO4*7H2O, pH7.0) at 37 °C to an OD600 of 0.6. Protein expression was induced by addition of 0.4 mM isopropyl ‐D‐thiogalactosidose (IPTG) at 30 °C for 3.5 h.
Bacteria were lysed in lysis buffer (20 mM sodium phosphate, pH 7.5, 150 mM NaCl, 40 mM Imidazol, 5 mM ‐ME, 1 tablet complete mini protease inhibitor® (Roche)/100 mL, 1 M Pepstatin A) in a Cell Disruptor at 2.5 kbar and 4 °C. 6xHis‐Sumo‐protein was purified by affinity chromatography using a HiTrapTM IMAC HP 5 mL column by the ÄKTATMexplorer system (GE Healthcare) controlled by the UNICORNTM software (GE Healthcare). 6xHis‐Sumo was cleaved
by subsequent affinity chromatography. Recombinant proteins were stored in Tris buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 5% glycerol) at ‐80 °C.
In Table 5 all used recombinant proteins are listed.
Table 5: Recombinant proteins used for in vitro assays.
Recombinant protein Source
3xFlag‐FAT10 Johanna Bialas, Kreuzlingen
6xHis‐VCP this work (plasmid provided by A. Buchberger) 6xHis‐VCPC this work (plasmid provided by A. Buchberger) 6xHis‐VCPN this work (plasmid provided by A. Buchberger) 6xHis‐VCPNC this work (plasmid provided by A. Buchberger) 6xHis‐VCP ND1 this work (plasmid provided by A. Buchberger) 6xHis‐VCP N only this work (plasmid provided by A. Buchberger) Diubiquitin, linear Enzo Life Science
FAT10 Nicola Catone, Kreuzlingen
Flag‐UBA6 Enzo Life Science
GST Dr. Annette Aichem, Kreuzlingen
GST‐FAT10 Dr. Annette Aichem, Kreuzlingen
His‐ISG15 Enzo Life Science
Sumo Enzo Life Science
Ulp1‐6xHis Nicola Catone, Kreuzlingen
VCP this work
4.9 In vitro assays
Because of impurities and degradation the real content of the actual protein in a preparation of recombinant proteins differs from the protein content measured by NanoDrop 1000 Spectrophotometer (Thermo Scientific). Thus for in vitro assays the amount was adjusted giving similar band intensities on coomassie gels. The stated amounts correspond to the measured values, but do not necessarily reflect real protein content.
For binding domain studies 6 g His‐VCP, 5.7 g His‐VCPC, 3.8 g His‐VCPN, 3.6 g His‐
VCPNC, 2.9 g His‐VCPND11.3 g His‐VCP N only, 20 g GST‐FAT10 and 5 g GST were incubated in Tris buffer (10 mM Tris, pH7.6, 25 mM NaCl, 5 mM MgCl2, 4 mM ATP, 0.1% (v/v) Triton X‐100, 1x Complete mini® protease inhibitor) for 30 min at 4 °C on a roller.
For in vitro FAT10ylation (Aichem, Catone et al. 2014) 0.5 g recombinant Flag‐UBA6 (Enzo Life Sciences) was used as E1 enzyme with an ATP recovery system (5 U/mL inorganic pyrophosphatase, 20 mM creatinphosphate, 4 g/mL creatinphosphokinase) with or without 4 mM ATP and incubated with 0.5 g VCP and 1.8 g FAT10 for 30 min at 30 °C in a shaker.
The colorimetric ATPase assay PiColorLockTM Gold (Innova Biosciences) was performed corresponding to the manufacturer’s protocol, including previous incubation with PiBindTM resin for 30 min at 4 °C on a roller to remove any residual phosphate. In a 96‐well plate 5 g recombinant VCP and 10 g 3xFlag‐FAT10 per well were incubated with 0.5 mM ATP and 5 mM MgCl2 containing 10 mM Tris buffer (pH7.5) at RT and the reaction was stopped every 5 min starting after 15 min up to 50 min using ‘Gold mix’. Samples were prepared in duplicates and read out in a SpectraFluor Plus microplate reader (Tecan) at 630 nm.
For the radioactive ATPase assay 1.25 g (12.8 nmol) recombinant VCP and increasing amounts of FAT10 or ULMs (molar ratio 0.2, 1, 5 and 10) were incubated in Tris buffer (10 mM Tris, pH7.5, 5 mM MgCl2) for 20 min on ice. ‐32P labelled ATP (9.25 MBq (250 Ci) activity;
12.5 mM; Hartmann Analytic) was diluted with the same Tris buffer to 1.25 mM. After addition of 1 L ATP, samples were incubated for 20 min at 37 °C. 1 L of the samples was spotted on to a Polygram© CEL 300 PEI thin layer chromatography (TLC) plate (Macherey‐Nagel) and separated by TLC in running buffer (0.7 M lithium chloride, 1 M acetic acid) for 20 min at RT.
Radioactivity was visualized by a Personal Molecular ImagerTM (PMI) system (Bio‐Rad). ATPase
4.10 Gel filtration chromatography
For analytical gel filtration chromatography recombinant proteins were concentrated up to 5‐
6 mg/mL and 1 mL containing either FAT10 or VCP alone or both together, preincubated for 30 min at 4 °C, was loaded onto a SuperdexTM 200 10/300 GL prep grade gel filtration column (GE Healthcare). Chromatography was performed with a Tris buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 5% (v/v) glycerol) by the ÄKTATMexplorer system (GE Healthcare). For determining the void volume blue dextran (2000 kDa) and for the standard curve the MWGF200 kit 12‐200 kDa (Sigma) containing cytochrome C (12.5 kDa), carbonic anhydrase (29 kDa), albumin (66 kDa), alcohol dehydrogenase (150 kDa) and ‐amylase (200 kDa) was used.