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

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Chapter 5 Figure 35: The antibody recognizes both monomeric FAT10 as well as FAT10 conjugates. HEK293T cells were transiently transfected with HA-FAT10 or left untreated and cell lysates were analyzed by western blot with a 100 ng/ml working dilution of clone 4F1. Both short as well as long exposures of the same blot are shown. A Ponceau staining of the membrane serves as a loading control. Materials and Methods Purification of recombinant proteins. The GST-FAT10 expression construct was generated as previously described by cloning the human FAT10 cDNA into pGEX-4T-3 (GE Healthcare) (Hipp et al., 2004). Recombinant proteins were purified in batch essentially according to the manufacturer’s instructions. The proteins used for coating ELISA plates were purified in the absence of Triton X-100. After purification, the buffer was exchanged to either PBS (for use in ELISA) or 0.9% (w/v) NaCl (for use in immunization) using a HiTrap Desalting 5ml or NAP-5 column (both GE Healthcare). Western blot, immunoprecipitation and immunofluorescence. Western blots, immunoprecipitations and immunofluorescences were performed as previously described (Kalveram et al., 2008). Expression constructs. HA-FAT10, HA-FAT10-GFP, HA-Ubiquitin-GFP, HA- SUMO-GFP (Hipp et al., 2005), HA-FAT10-N-GFP, HA-FAT10-C-GFP (Schmidtke et al., 2006) as well as GST-FAT10, FLAG-NEDD8 (Hipp et al., 2004) and FLAG- 117
Chapter 5 Figure 36: The antibody specifically recognizes the N-terminal UBL domain of FAT10. Lysates of HEK293T cells transiently transfected with the indicated constructs or treated with IFN-γ and TNF-α were analyzed by western blot with (A) either a 50 ng/ml working dilution of clone 4F1 or (B) anti-GFP or anti-FLAG antibodies as a loading control. 118
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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
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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 and 116: Chapter 5 binant human and mouse GS
Page 117: Chapter 5 Figure 34: The antibodies
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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