Role of the ubiquitin-like modifier FAT10 in protein degradation and ...

Chapter 2
centrifuged, the supernatant was removed, and the beads were washed with Tev-
buffer. The supernatant and the wash were combined and diluted in 50 mM Tris-
HCl (pH 7.5), 2 mM DTT, 5 mM MgCl2, 20% glycerol. The DTT was removed and
the protein was concentrated by ultrafiltration. The Tev-protease was removed
by Ni ++ -affinity chromatography as suggested by the supplier (Invitrogen).
Preparation of radiolabeled in vitro transcribed/translated ODC was performed
by a T7 polymerase-dependent transcription-translation (TNT)-coupled system
from reticulocyte lysate (Promega) in the presence of TranS 35 label as recom-
mended by the supplier. Following the translation reaction, unincorporated [ 35 S]-
labeled methionine/cysteine was removed and the buffer was exchanged to 50 mM
Tris-HCl (pH 7.5), 2 mM DTT, 5 mM MgCl2, 20% glycerol by gel filtration chro-
matography on a NICK column (GE healthcare). The protein was concentrated
by ultrafiltration in a Centricon 30 unit. Specific activity for [ 35 S]-labeled pro-
teins was estimated to be about 5 × 10 4 c.p.m./pmol. After buffer exchange to
50 mM Tris-HCl pH 7.5, 10 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM ATP and
20% glycerol, the concentration of labeled proteins was estimated by SDS-PAGE
and coomassie blue staining, using unlabeled proteins as a standard.
In vitro degradation assays. Protein degradation assays were performed
as previously outlined (Ben-Shahar et al., 1999; Hoyt et al., 2006) with some
modifications. The assays were performed in 50 µl reaction volumes at 37°C
for one hour and contained 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM ATP,
20% glycerol, an ATP regenerating system (2 mM dithiothreitol, 10 mM creatine
phosphate, 1.6 mg/ml creatine kinase), 1 mg/ml acetylated bovine serum albumin
(Promega) and proteasomes. Reactions were initiated by addition of substrate
(usually 20000cpm) and quenched by addition of 50 µl of 20% (w/v) trichloroacetic
acid. The trichloroacetic acid-insoluble material was removed by centrifugation
(14,000 × g, 15 min) at 4°C, and 3 × 30 µl of the supernatant was removed and re-
leased counts were measured in a scintillation counter. Percentage degradation of
radiolabeled proteins was determined as the released counts/min divided by total
input in counts/min. Total input counts were determined by substituting water
for trichloroacetic acid in the reactions. Background counts/min were determined
in reactions devoid of proteasomes, and were typically 1% to 3% of total input
counts.
Western blot and immunoprecipitation. Determination of NUB1L protein
expression was performed by Western blotting using anti-NUB1L peptide anti-
bodies provided by Biomol and Dr. Michal E. Cheetham (London) (van der Spuy
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