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

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Chapter 5 Figure 33: Immunofluorescence of hybridoma supernatants. HEK293T cells were grown on poly-L-lysine-coated coverslips, transiently transfected with human HA-FAT10 and treated with 5µM MG132 or solvent only for 6 hours. Cells were then fixed and stained with either an alexa-488-coupled anti-HA antibody (green) or 3A11 or 4F1 hybridoma supernatants followed by an alexa-546-coupled secondary anti-mouse antibody (red) as well as DAPI (blue). Untransfected cells serve as the negative control. Scale bar = 5µm. The monoclonal anti-FAT10 antibody recognizes both monomeric FAT10 as well as FAT10-conjugates As both hybridomas most likely produce the same antibody, the optimal working dilution was only titrated for one of the clones (4F1) and was determined to lie between 50 and 100 ng/ml. HEK293T cells were either left untreated or transiently transfected with HA-FAT10 and cell lysates as well as 1:2 and 1:10 dilutions of the HA-FAT10 lysates were analyzed by western blot with a 100 ng/ml dilution of anti-FAT10 (clone 4F1) antibody in 3% BSA. Both short and long exposures of the same blot are shown in figure 35 and the longer exposure reveals the presence of high molecular weight FAT10-conjugates in the cells transfected with HA-FAT10. A Ponceau staining of the membrane serves as a loading control. 115
Chapter 5 Figure 34: The antibodies produced by both hybridomas are able to immunoprecipitate FAT10. HEK293T cells were transiently transfected with HA-FAT10 or left untreated and the cell lysates were subjected to immunoprecipitation with 5 µg of either anti- HA antibody, clone 3A11 or clone 4F1. The precipitated proteins as well as the lysates before (input) as well as after (supernatants) the IP were analyzed by anti-HA western blot. The monoclonal anti-FAT10 antibody specifically recognizes the N-terminal UBL domain of FAT10 To acquire information about the specificity of the antibody and the localization of the epitope recognized by it, HEK293T cells were transiently transfected with constructs containing N-terminal fusions of the isolated UBL domains or full- length FAT10 to GFP. Several other ubiquitin-like modifiers, namely ubiquitin- GFP, SUMO-GFP, LC3B-GFP and FLAG-NEDD8 were also transfected. In addition, expression of endogenous FAT10 was induced by treatment of cells with IFN-γ and TNF-α. Lysates of these cells were then analysed by western blot with either anti-GFP or anti-FLAG antibodies or a 50 ng/ml dilution of clone 4F1. Figure 36 reveals that the monoclonal anti-FAT10 antibody recognized both full- length FAT10 as well as its N-terminal UBL domain. In addition, it demonstrates that this antibody did not cross-react with any of the other ubiquitin-like modifiers investigated except for LC3 (ATG8). Interestingly, LC3 is the only one among the proteins assayed for cross-reactivity which shares no sequence homology with FAT10. 116
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Role of the ubiquitin-like modifier
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Acknowledgements . . . . . . . . .
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Summary / Zusammenfassung English F
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Summary In conclusion, these result
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Summary nicht von der katalytischen
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The Ubiquitin-Proteasome-System The
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Introduction Figure 1: The 26S prot
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Introduction are the major source o
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Introduction majority of ubiquitin-
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Introduction example contained in t
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Introduction two modifiers apart fr
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Introduction Figure 4: Comparison o
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Introduction dependent apoptosis in
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Introduction Interestingly, a recen
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The Autophagy-Lysosome System Intro
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Introduction Figure 5: Molecular ma
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Introduction The transport of misfo
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Introduction where the proteasome i
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Chapter 1 The UBA domains of NUB1L
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Introduction Chapter 1 Ubiquitin, a
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Chapter 1 no correlation between th
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Chapter 1 covered proteins were sep
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Chapter 1 Figure 8: Accelerated deg
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Chapter 1 Treatment with IFN-γ and
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Chapter 1 ing SUMO-1-GFP or the C-t
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Chapter 1 Figure 12: Accelerated de
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Chapter 1 Figure 13: Co-immunopreci
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Chapter 1 domains of NUB1L is requi
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Chapter 1 Rad23 (Verma et al., 2004
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Chapter 1 FAT10-GFP expressing vect
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Chapter 2 Degradation of FAT10 by t
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Introduction Chapter 2 The proteaso
Page 65 and 66: Chapter 2 From our previous data it
Page 67 and 68: Chapter 2 Figure 14: Purified compo
Page 69 and 70: Chapter 2 evaluation of the NUB1L b
Page 71 and 72: Chapter 2 teins which contain intri
Page 73 and 74: Chapter 2 Combined with our studies
Page 75 and 76: Chapter 2 Recombinant expression of
Page 77 and 78: Chapter 2 et al., 2003), both at a
Page 79 and 80: Abstract Chapter 3 During misfolded
Page 81 and 82: Chapter 3 tion between the UPS and
Page 83 and 84: Chapter 3 Figure 18: HDAC6 interact
Page 85 and 86: Chapter 3 Figure 19: Both the BUZ d
Page 87 and 88: Chapter 3 of HDAC6 was required for
Page 89 and 90: Chapter 3 precipitates with HDAC6 (
Page 91 and 92: 90 Chapter 3
Page 93 and 94: Chapter 3 Figure 23: The model conj
Page 95 and 96: Chapter 3 HDAC6 is required for the
Page 97 and 98: Chapter 3 Figure 26: Both ubiquitin
Page 99 and 100: Chapter 3 also revealed endogenous,
Page 101 and 102: Chapter 3 and/or its conjugates is
Page 103 and 104: Chapter 3 (Hipp et al., 2005), pEGF
Page 105 and 106: Chapter 4 FAT10-deficient mice disp
Page 107 and 108: Chapter 4 Once the infection is cle
Page 109 and 110: Chapter 4 As both wild-type as well
Page 111 and 112: Chapter 4 Peptides. The synthetic p
Page 113 and 114: Introduction Chapter 5 Antibodies a
Page 115: Chapter 5 binant human and mouse GS
Page 119 and 120: Chapter 5 Figure 36: The antibody s
Page 121 and 122: Discussion 30 years ago, ubiquitin
Page 123 and 124: Discussion monomeric ubiquitin as w
Page 125 and 126: Discussion Alternatively, FAT10 cou
Page 127 and 128: References D. T. Akey, X. Zhu, M. D
Page 129 and 130: References Y.-H. Chiu, Q. Sun, and
Page 131 and 132: References S. Gunawardena, L.-S. He
Page 133 and 134: References J. A. Johnston, M. E. Il
Page 135 and 136: References F. Lévy, N. Johnsson, T
Page 137 and 138: References J.-H. Park, H.-S. Jong,
Page 139 and 140: References P. Schreiner, X. Chen, K
Page 141 and 142: References S. Vijay-Kumar, C. E. Bu
Page 143 and 144: Abbreviations aa amino acid AAA ATP
Page 145 and 146: Amino Acids Alanine Ala A Arginine
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