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

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Chapter 5 Generation of mouse monoclonal antibodies directed against human FAT10 Birte Kalveram, Gunter Schmidtke and Marcus Groettrup Unpublished results. 111
Introduction Chapter 5 Antibodies are invaluable tools for the study of endogenous proteins and can be utilized for example for the determination of tissue specific expression patterns through western blot or histology or the analysis of intracellular localization by immunofluorescence. We have previously generated two polyclonal antibodies by immunizing rabbits with either recombinant GST-FAT10 or KLH-coupled synthetic peptides (Hipp et al., 2005; Raasi, 2001), which have been successfully used in a number of applications such as western blotting, immunoprecipitation and immunofluorescence. Unfortunately, use of the GST-FAT10 antiserum has been somewhat limited by its high level of cross-reactivity and we therefore decided to generate monoclonal antibodies directed against human FAT10 through the immunization of mice with recombinant GST-FAT10. Furthermore, the availability of two antibodies derived from different species will enable us to perform a number of additional applications, such as for example the detection of low levels of FAT10 protein in different tissues through the combination of immunoprecipitation and western blot. Results and Discussion Since a previous attempt to generate FAT10-specific monoclonal antibodies in BALB/c mice failed to yield any viable hybridomas, we decided to use FAT10- deficient mice for the immunization in the hope that these would carry a higher number of FAT10-specific precursor cells. Two mice were immunized with recombinant human GST-FAT10 and after three rounds of booster immunizations displayed a titer high enough to warrant performing the fusion. Unfortunately, immunizations had to be carried out with a GST-fusion protein as proteolytic re- moval of GST resulted in the immediate precipitation of untagged FAT10. Sim- ilarly, expression of FAT10 with a smaller tag (e.g. His6) did not yield any sol- uble protein either. To exclude those hybridomas which produced antibodies directed against GST, hybridoma supernatants were screened by ELISA for reactivity against both GST-FAT10 as well as GST alone. The initial screening yielded five positive clones, two of which could be stabilized after two (in the case of 4F1) or three (in the case of 3A11) rounds of subcloning. Isotyping by ELISA revealed both antibodies to be of the IgG2a isotype. 112
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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
Page 61 and 62: 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 (
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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: Chapter 4 Peptides. The synthetic p
Page 115 and 116: Chapter 5 binant human and mouse GS
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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