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

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Chapter 5 The antibodies produced by both clones recognize transiently transfected and endogenous FAT10 To determine whether the antibodies were able to recognize FAT10 in a western blot, HEK293T cells were transiently transfected with FLAG-FAT10, treated with 500 U/ml hTNF-α and 200 U/ml hIFN-γ for 16 hours or left untreated. The cell lysates were subsequently analyzed by western blot with a 1:1000 dilution of the purified antibodies in 3% BSA, which resulted in a 6.8 µg/ml (3A11) or 4.4 µg/ml (4F1) working dilution, respectively. Both antibodies were able to recognize FAT10 equally well and the signal strength was comparable to that of an anti-FLAG western blot of the same lysates (Fig. 31). Interestingly, the antibodies from both hybridomas produce in the same background bands, are of the same isotype and recognize the N-terminal UBL domain of FAT10 (Fig. 36 and data now shown). This suggests that, even though both hybridomas are independent clones, they probably produce the same antibody. Figure 31: The antibodies produced by both hybridomas recognize ectopically expressed as well as endogenous FAT10 in a western blot. HEK293T cells were transiently transfected with human FLAG-FAT10, treated with IFN-γ and TNF-α or left untreated and cell lysates were analyzed by western blot with either anti-FLAG antibody (right panel) or clone 3A11 (left panel) or clone 4F1 (middle panel) at working dilutions of 4.4µg/ml and 6.8µg/ml, respectively. Neither antibody is able to recognize murine FAT10 In order to determine whether the antibodies were perhaps also capable of recognizing murine FAT10 in addition to the human protein, equal amounts of recom- 113
Chapter 5 binant human and mouse GST-FAT10 were loaded onto an SDS-PAGE and subsequently probed with a 1:1000 dilution of the purified antibodies in 3% BSA. Un- fortunately, neither clone 3A11 nor clone 4F1 was capable of recognizing murine FAT10 in a western blot (Fig. 32). Figure 32: The antibodies produced by both hybridomas recognize only human and not murine FAT10. Clones 3A11 (left panel) and 4F1 (middle panel) at working dilutions of 4.4µg/ml and 6.8µg/ml, respectively, were tested for reactivity with recombinant human (GST-hFAT10) as well as mouse (GST-mFAT10) FAT10 by western blot. A blot probed with anti-GST antibody (Sigma) serves as the loading control (right panel). The antibodies produced by both hybridomas recognize FAT10 in immunofluorescence To determine whether the antibodies could be used to detect FAT10 in immunofluorescence stainings, HEK293T cells were grown on poly-L-lysine-coated cover slips, transiently transfected with HA-FAT10, fixed with 4% paraformaldehyde and stained with either anti-HA antibody or either 3A11 or 4F1 hybridoma su- pernatant which was first concentrated approximately 50-fold and subsequently diluted 1:5 in 1.5% normal goat serum. As can be seen in figure 33, the antibodies produced by both clones recognize FAT10 equally well and with little background in immunofluorescence. The antibodies produced by both clones are able to immunoprecipitate FAT10 HEK293T cells were transiently transfected with HA-FAT10 or left untreated and their lysates were subjected to an immunoprecipitation with 5 µg of either anti- HA antibody or anti-FAT10 clone 3A11 or clone 4F1. The precipitates as well as the lysates before and after the IP were subsequently analyzed by western blot with an anti-HA antibody. As can be seen in figure 34, the antibodies produced by both clones are able to precipitate HA-FAT10 with the same efficiency as an anti-HA antibody. 114
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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
Page 63 and 64: 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: Introduction Chapter 5 Antibodies a
Page 117 and 118: Chapter 5 Figure 34: The antibodies
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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