Role of the ubiquitin-like modifier FAT10 in protein degradation and ...
Role of the ubiquitin-like modifier FAT10 in protein degradation and ...
Role of the ubiquitin-like modifier FAT10 in protein degradation and ...
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Chapter 1<br />
covered prote<strong>in</strong>s were separated by SDS-PAGE <strong>and</strong> analysed by autoradiogra-<br />
phy. As can be seen <strong>in</strong> figure 7A, C <strong>and</strong> 9C, only wild-type NUB1L, NUB1, <strong>and</strong><br />
NUB1L∆UBL were able to pull down His6-<strong>FAT10</strong> from <strong>the</strong> lysates. A subsequent<br />
immunoprecipitation <strong>of</strong> <strong>the</strong> supernatants with anti-His6 antibody demonstrated<br />
that sufficient His6-<strong>FAT10</strong> was available <strong>in</strong> all reactions, so lack <strong>of</strong> expression can<br />
be excluded as a reason for <strong>the</strong> negative results. From <strong>the</strong> experiments presented<br />
<strong>in</strong> figure 6, figure 7 <strong>and</strong> figure 9C, D we conclude that <strong>the</strong> <strong>in</strong>teraction <strong>of</strong> <strong>FAT10</strong><br />
<strong>and</strong> NUB1L depends on <strong>the</strong> presence <strong>of</strong> all three UBA-doma<strong>in</strong>s, but is <strong>in</strong>depen-<br />
dent <strong>of</strong> <strong>the</strong> UBL-doma<strong>in</strong> <strong>of</strong> NUB1L. The deletion <strong>of</strong> only 14 am<strong>in</strong>o acids <strong>in</strong> NUB1,<br />
which is about one third <strong>of</strong> <strong>the</strong> UBA2 doma<strong>in</strong> is not sufficient to abrogate <strong>the</strong><br />
<strong>in</strong>teraction <strong>of</strong> NUB1 <strong>and</strong> <strong>FAT10</strong>.<br />
Figure 7: Co-immunoprecipitation <strong>of</strong> NUB1L mutants <strong>and</strong> <strong>FAT10</strong>. (A, C)Empty vector<br />
(mock) or HA-tagged NUB1L mutants lack<strong>in</strong>g one or more UBA-doma<strong>in</strong>s were transfected<br />
ei<strong>the</strong>r alone (- His-<strong>FAT10</strong>) or toge<strong>the</strong>r with His 6-tagged <strong>FAT10</strong> (+ His-<strong>FAT10</strong>) <strong>in</strong>to<br />
HEK293T cells. The cells were lysed <strong>and</strong> an anti-HA immunoprecipitation was performed.<br />
The prote<strong>in</strong>s bound by <strong>the</strong> antibody were separated by SDS-PAGE <strong>and</strong> analyzed<br />
by autoradiography. The NUB1L mutants appear between <strong>the</strong> 60 <strong>and</strong> <strong>the</strong> 90<br />
kD molecular weight markers <strong>in</strong>dicated at <strong>the</strong> right. An arrow on <strong>the</strong> right side <strong>of</strong> <strong>the</strong><br />
gel denotes <strong>the</strong> position <strong>of</strong> co-immunoprecipitated <strong>FAT10</strong>. Only wild-type HA-NUB1L<br />
<strong>and</strong> HA-NUB1L∆UBL coprecipitated significant amounts <strong>of</strong> His 6-<strong>FAT10</strong>. (B, D) To analyze<br />
<strong>the</strong> amount <strong>of</strong> <strong>FAT10</strong> available, <strong>the</strong> supernatants after <strong>the</strong> immunoprecipitation<br />
shown <strong>in</strong> A <strong>and</strong> C were precipitated with anti-His 6 antibodies, <strong>and</strong> <strong>the</strong> bound prote<strong>in</strong>s<br />
separated by SDS-PAGE. Lane occupation is <strong>the</strong> same as <strong>in</strong> A <strong>and</strong> C, respectively. His 6-<br />
<strong>FAT10</strong> was present <strong>in</strong> all <strong>FAT10</strong> transfected cells. The experiments were repeated three<br />
times yield<strong>in</strong>g similar results.<br />
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