Images for working group survey

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Images to support working group survey

These are images to support the GO camp working group survey, available at:

'Response to' Working Group Question I

PMID: 19919618 Identification of stress-related proteins in Escherichia coli using the pollutant cis-dichloroethylene.

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Binding Working Group Question I

PubMed: 18636086 The tumour suppressor CYLD is a negative regulator of RIG-I-mediated antiviral response

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Fig.3A: Co-immunoprecipitation experiments of Myc-tagged CYLD and Flag-tagged RIG-I, IPS-1, TBK1 or IKKe in 293 EBNA cells. When the stringency of the wash was increased to 500mM NaCl, the interaction of TBK1 or IKKe with CYLD was no longer observed.

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Fig 3C: EBNA cells were transfected with Flag-tagged RIG-I, RIG-IN, IPS-1, TBK1 or IKKe, together with HA-tagged K63-Ub and either empty vector or Myc-tagged CYLD. Lysates were denatured in 1% SDS and immuno-precipitated with anti-Flag. The immunoprecipitations were blotted with anti-HA to detect K63-Ub and re-probed with anti-Flag. The input lysates were blotted with anti-Myc to detect CYLD

'Regulation' Working Group Question I

PMID: 12783775 Inhibition of Foxo1 function is associated with improved fasting glycemia in diabetic mice.

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Fig. 1. Effects of production of the mutant of forkhead transcription factor Foxo1 (devoid of its carboxyl domain, Foxo1- 256), on gluconeogenic gene expression in H4IIE cells. A: adenoviral mediated production of Foxo1- 256 in H4IIE cells. Arrow indicates the protein band of Foxo1- 256 in a 12% SDS-polyacrylamide gel, as visualized by Western blot analysis. MOI, multiplicity of infection. B–D: Northern blot analysis of phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G-6-Pase), and Foxo1- 256 mRNA. Endogenous PEPCK (B), G-6-Pase (C), and Foxo1- 256 (D) gene expression in H4IIE cells were studied by Northern blot analysis and quantified by PhosphorImager using GAPDH mRNA as an internal control. Data were obtained from 5, 3, and 2 independent measurements for PEPCK, G-6-Pase, and Foxo1- 256 mRNAs, respectively. E: intracellular levels of caspase-3 activity. F: genomic DNA integrity of the Foxo1- 256 and control vector-transduced H4IIE cells. MW, molecular weight. *P < 0.001 vs. control values, by ANOVA.

'Downstream effects' Working Group Question I

PMID:20194890 CYLD regulates angiogenesis by mediating vascular endothelial cell migration" Gao et al. 2010

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Figure 3. CYLD regulates angiogenesis by mediating cell spreading and migration. (A) HUVECs transfected with pEGFPC1 and pSUPER or pSUPER-CYLD plasmids for 72 hours were plated onto matrigel, and photographs were taken 25 minutes later. White arrowheads indicate cells spreading on the plates, and black arrowheads indicate cells without such a property. Objective lens used was A-Plan 20x/0.3 NA dry (Carl Zeiss Inc). (B) Experiments were performed as in panel A, and the degree of cell spreading was quantified 25 and 60 minutes after plating. Data are the mean and SE from 2 experiments (600 transfected cells were measured for each experiment; **P < .01 vs control). (C) HUVECs transfected with CYLD or control siRNAs for 72 hours were scratched, and wound margins were imaged 12 hours later. Objective lens used was A-Plan 20x/0.3 NA dry (Carl Zeiss Inc). (D) Experiments were performed as in panel C, and the extent of wound closure was quantified by measuring the wound area compared with the initial wound area. Data are the mean and SE from 2 experiments with each performed in triplicate (**P < .01 vs control). (E) HUVECs were transfected with pEGFPC1 and pSUPER or pSUPER-CYLD plasmids, and the fluorescence of GFP at the leading edge of cells was recorded at 20-second intervals with the use of the TCS SP5 confocal microscope (Leica), equipped with a live-cell imaging workstation and the LAS AF software. Rectangular regions were selected as indicated to analyze membrane ruffle dynamics. Objective lens used was HCX Plan-Apochromat 20x/0.7 NA dry (Leica). (F) Experiments were performed as in panel E, and membrane ruffle dynamics were presented as 3-dimensional surface plots.

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Figure 4. CYLD modulates microtubule dynamics and cell polarization in migrating cells. (A) HUVECs were transfected with pEGFPC1– -tubulin and pSUPER or pSUPER-CYLD, and the dynamics of individual GFP-labeled microtubules in migrating cells were then recorded with the TCS SP5 confocal microscope (Leica), equipped with a live-cell imaging workstation and the LAS AF software. Objective lens used was HCX Plan-Apochromat 63x/1.4 NA oil (Leica). (B) Experiments were performed as in panel A, and individual microtubule life history plots representing changes in microtubule length over time were generated. Growth events exhibit an increase in distance from a fixed point (y-axis) over time (x-axis), and shortening events are seen as a decrease in distance over time. (C) HUVECs transfected with CYLD or control siRNAs were scratched, and cells were fixed 2 hours later and stained with anti– -tubulin antibody, anti–pericentrin antibody, and DAPI to visualize microtubules (green), centrosomes (red), and nuclei (blue), respectively. Broken white lines indicate the wound direction. Objective lens used was Plan-Apochromat 63x/1.25 NA oil (Carl Zeiss Inc).

'Response to' Working Group Question II

PMID: 11847130 "ERp44, a novel endoplasmic reticulum folding assistant of the thioredoxin family." Anelli et al. 2002

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Fig. 3. ERp44 transcripts are widely expressed in human cell lines and can be further induced by ER stress. (A) Northern blot analyses were performed on total RNA extracted from the cell lines indicated, obtained from ATCC. Blots were hybridized sequentially with probes specific for ERp44 (top panel) or GAPDH (bottom panel) for normalization. (B) ERp44 transcripts accumulate during the UPR. SK-N-BE neuroblastoma cells were treated with tunicamycin (TM, 10 µg/ml), DTT (2 mM) or both drugs for 6 h (Benedetti et al., 2000). Blots were hybridized with probes specific for ERp44, actin and BiP, as indicated. Densitometric quantitations are reported on the right, and expressed as fold induction relative to untreated cells.

'Regulation' Working Group Question II

PMID: 18536048 "Peroxisomal acyl-CoA-oxidase deficiency: two new cases." Carrozzo R et al.

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'Binding' Working Group Question II

PubMed: 16472752 Crystal Structures of U8 snoRNA Decapping Nudix Hydrolase, X29, and Its Metal and Cap Complexes

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Fig. C: Dependence of decapping activity on [MnCl2] and the Hill plot (inset) derived from this binding activity curve

'Downstream Effects' Working Group Question II

PMID:16337599 The human homolog of yeast BRE1 functions as a transcriptional coactivator through direct activator interactions. Kim et al. 2005

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Figure 2. hBRE1 Specifically Increases the Global Level of H2B Ubiquitylation at Lysine 120 (A–C) FLAG-H2B was coexpressed with yRAD6, yBRE1, and hBRE1 (A), indicated combinations of hBRE1 and HA-ubiquitin (B), and indicated combinations of hE1, hBRE1, and hRAD6B (C) in 293T cells. Cell extracts were immunoprecipitated with M2 agarose and subjected to immunoblot analysis with anti-FLAG and anti-HA (Figure 2B, bottom) antibodies. Immunoblot of whole-cell extracts for hBRE1 and GAPDH (Figure 2C, middle and bottom, respectively). (D and E) FLAG-core histones (D) and FLAG-H2B and the H2BK120R mutant (E) were transiently expressed in the presence or absence of hBRE1 in 293T cells, as indicated, and analyzed as in Figure 2A.

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Figure 7. hBRE1 Is Linked to Histone H3-K4 and -K79 Methylation (A–D) 293T cells were transiently transfected with control, hE1, hBRE1, or hRAD6B expression plasmids (A), and 293T cells were treated with siRNA targeting nonspecific (control) and hBRE1 as indicated (B). Total cell lysates were subjected to immunoblot analysis with antibodies as indicated. The proteins levels in (A) (shown in [C]) and in (B) (shown in [D]) were normalized to H3 and scored respectively.