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

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Chapter 4 derived dendritic cells (BMDCs) argue against a role of FAT10 in the death of DCs, as BMDCs derived from FAT10-deficient as well as wild-type mice displayed no differences in the rate of maturation-induced apoptosis (data not shown). How- ever, as BMDCs constitute a highly artificial system, these experiments will need to be repeated with spleenic DCs for a definite answer. In addition, spleenic T cells could be assayed for differences in apoptosis after bulk stimulation through recep- tor cross-linking with anti-CD3 and anti-CD28 antibodies and subsequent restim- ulation with either anti-CD3 antibodies alone, treatment with PMA/ionomycin or FasL to induce ACID; or they could be cultivated in cytokine-free medium to induce ACAD. Materials and Methods Mice. FAT10 gene-targeted mice were generated as previously described (Canaan et al., 2006) and were backcrossed on the C57BL/6 background until the 10 th gen- eration was obtained. Genotyping of mice was performed as described previously, except for an alternative set of primers which was used to amplify the neomycin gene: NeoF2 (5’-TATCGCCTTCTTGACGAGTTC-3’) and NeoR2 (5’-CATCAGGTG GAGTCTCTCTC-3’). C57BL/6 mice (H-2 b ) mice were obtained originally from Charles River Laboratories. Mice were kept in a specific pathogen-free facility and used at 6 - 10 weeks of age. Animal experiments were approved by the Regierungspräsidium Freiburg. Virus. LCMV-WE was obtained originally from F. Lehmann-Grube (Hamburg, Germany) and propagated in the fibroblast line L929. Mice were infected with a low dose of 200 pfu of LCMV-WE i.v., and the specific CTL response was analyzed at days 7, 14 and 20 post infection. Intracellular staining (ICS) for IFN-γ. Splenocytes (2 × 10 6 ) were incubated in round-bottom 96-well plates with 10 -7 M of the specific peptide in 100 µl medium containing 10 µg/ml brefeldin A for 5 h at 37°C. The staining, fixation, and per- meabilization of the cells were performed exactly as previously described (Basler et al., 2004). Briefly, cells were first stained with an anti-CD8-Tri-color conjugate (Caltag) at 4°C followed by fixation in 4% paraformaldehyde. Cells were then permeabilized in 0.1% saponin, stained with an anti-IFN-γ-FITC conjugate (BD Biosciences) and analyzed in a FACScan flow cytometer. 109
Chapter 4 Peptides. The synthetic peptides GP33–41 (KAVYNFATC), GP92–101 (CSANNS- HHYI), GP118–125 (ISHNFCNL), GP276–286 (SGVENPGGYCL), NP205–212 (YTVKY- PNL), and NP396–404 (FQPQNGQFI) were obtained from Echaz Microcollections. BMDCs. Bone marrow-derived dendritic cells were generated as previously described (Schlosser et al., 2008). Briefly, bone marrow cells from FAT10 -/- as well as C57BL/6 mice were cultured in the presence of GM-CSF for seven days. Af- ter differentiation, non- and semiadherent cells were harvested and treated wih 1µg/ml LPS to induce maturation. 24 and 48 hours later, cells were stained with FITC-coupled annexin-V and propidium iodide (both BD Biosciences) according to the manufacturer’s instructions and analyzed for cell death in a FACScan flow cytometer. 110
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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
Page 59 and 60: 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 (
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: Chapter 4 As both wild-type as well
Page 113 and 114: Introduction Chapter 5 Antibodies a
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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