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1.
Figure 1

Figure 1. From: Defining human ERAD networks through an integrative mapping strategy.

Hierarchical cluster analysis of CompPASS-identified HCIPs. Hierarchical clustering of HCIPs for interactions present in DIG (left) and TX-100 (right). Prominent HCIP clusters identified in DIG (1–8D) and TX-100 (1–3, 5 and 8T) were manually selected and are highlighted below. Box colour indicates the WDN-score.

John C. Christianson, et al. Nat Cell Biol. ;14(1):93-105.
2.
Figure 5

Figure 5. From: Defining human ERAD networks through an integrative mapping strategy.

Coordinated ER stress response of ERAD genes. qRT-PCR results for validated and suspected ERAD components upon treatment of HEK293 cells with TUNIC (10μg/mL, 6 hr). Data are presented as fold induction (log2) normalised to β-Actin. TUNIC-induced expression changes in ERAD genes plotted as groups according to: (a) Fold induction of gene expression represented by functional category. (c) Fold induction of gene expression from panel (b) mapped onto the ERAD interactome from . Additional genes of interest are presented alongside the induction map.

John C. Christianson, et al. Nat Cell Biol. ;14(1):93-105.
3.
Figure 3

Figure 3. From: Defining human ERAD networks through an integrative mapping strategy.

shRNA-mediated refinement of Hrd1 complex interactions. (a–q) S-tagged ERAD baits were transiently coexpressed with the indicated shRNAs in HEK293 cells. All complexes were affinity-purified (AP) in 1% DIG and analysed by immunoblotting. (a) XTP3-B-S expression, probe for Hrd1 & SEL1L simultaneously; (b) Coexpression of myc-UBE2J1 and XTP3-B-S, probe for myc & SEL1L; (c) XTP3-B-S expression, probe for FAM8A1 & SEL1L; (d) S-OS-9 expression, probe for Hrd1 & SEL1L simultaneously; (e) Incorporation of refinements (a–d) to the Hrd1 complex; (f) S-FAM8A1 expression, probe for Hrd1 & SEL1L; (g) Coexpression of myc-UBE2J1 and Hrd1-S, probe for myc & SEL1L; (h) Coexpression of myc-UBE2J1 and S-SEL1L, probe for myc & Hrd1; (i) AUP1-S expression, probe for Hrd1 & SEL1L; (j) Coexpression of myc-UBE2G2, AUP1-S pulldown, probe for myc & Hrd1; (k) Refined interaction map for Hrd1 complex. (l) UBAC2-S expression with UBXD8 knockdown, probe for gp78; (m) UBAC2-S expression with gp78 knockdown, probe for UBXD8; (n) UBXD8-S expression with gp78 knockdown, probe for UBAC2; (o) UBXD8-S expression with UBAC2 knockdown, probe for Derlin-2, UBAC2, & gp78; (p) UBXD8-S expression with Derlin-2 knockdown, probe for gp78; (q) Refined interaction map for gp78 complex.

John C. Christianson, et al. Nat Cell Biol. ;14(1):93-105.
4.
Figure 2

Figure 2. From: Defining human ERAD networks through an integrative mapping strategy.

The Interaction Network for ERAD (INfERAD). Interaction network for ERAD isolated in DIG and TX-100 represented by baits (squares) and their HCIPs (circles). Unidirectional (short dash, single arrow) and reciprocal (solid black, double arrows) interactions are shown. Each bait protein is rendered in a unique colour and line colour reflects the bait protein used to identify the interaction with the HCIP. Short dashed lines marked with a circle indicate interactions detected in both DIG and TX-100, while long dashed lines represent those found only in TX-100. The inset table lists the determined constituents of the mEMC, their size, cellular localisation, and corresponding yeast orthologs (SC) and ID in the Saccharomyces Genome Database (SGD). For clarity, a selection of additional DIG HCIPs not included in the map is shown on the bottom, with a circle’s colour corresponding to the bait for which the HCIP was observed and asterisks denoting an HCIP also detected in TX-100.

John C. Christianson, et al. Nat Cell Biol. ;14(1):93-105.
5.
Figure 8

Figure 8. From: Defining human ERAD networks through an integrative mapping strategy.

Functional integration of mammalian ERAD networks. The schematic model of the ERAD protein interaction network is topologically organised with respect to the ER membrane and arranged as an array of 6 colour-coded functional modules. Individual components from this study (baits or HCIPs) are indicated as nodes with reported components (black) and novel components (red). Similarly, reported interactions confirmed in this study (black) and novel interactions (red) are shown. Symbols for protein-protein interactions, UPR induction, and functional requirements are indicated in the legend. Intermodule interactions represented terminate either at the specific node within a module that establishes the link with the module periphery or at the module itself (where there are interactions with multiple components and that module is a single complex, (e.g. the mEMC or proteasome)). Asterisks indicate components that were identified by proteomics, but exhibited a subthreshold CompPASS score (WDN-score < 1.0).

John C. Christianson, et al. Nat Cell Biol. ;14(1):93-105.
6.
Figure 7

Figure 7. From: Defining human ERAD networks through an integrative mapping strategy.

Characterisation of UBAC2, a novel ubiquitin-binding ERAD component. (a) Predicted domain structure and interaction network of UBAC2. (b) Immunoprecipitation with anti-UBXD8 from HEK293 DIG soluble lysates were analysed by Western blotting with the indicated antibodies. (c) Analysis of muliple UBAC2 targeting shRNAs on the Hrd1 substrate TTR(D18G)-GFP by flow cytometry. (d) Sequence alignment of the predicted UBA domains from UBAC2 (304–344) and UBXD8 (8–53) with characterised human and yeast UBA domains. (e) HeLa cells expressing C-terminally S-tagged UBAC2 or gp78 were permeablised, immunostained, and analysed by fluorescence microscopy as in . (f) Recombinantly expressed UBA domains of hPlic2, UBXD8, and UBAC2 were coupled to Affigel and incubated with HEK293 cell lysates (−/+ 10 μM MG132, 6 hr). Samples were separated by SDS-PAGE, and ubiquitin binding was determined by immunoblotting with anti-Ub.

John C. Christianson, et al. Nat Cell Biol. ;14(1):93-105.
7.
Figure 4

Figure 4. From: Defining human ERAD networks through an integrative mapping strategy.

Functional genomic screen to identify essential substrate-specific ERAD components. (a) Localisation and topology of GFP reporters; TTR(D18G)-GFP, A1AT(NHK)-GFP, A1AT(NHKQQQ)-GFP, GFP-GluR1, GFP-CFTR(ΔF508), and GFPu, and GFP. (b) Time course of relative mean GFP fluorescence levels for each ERAD reporter cell line treated with MG132 (10 μM). (c) Heat maps reflecting the normalised fold change in mean GFP fluorescence of ERAD reporter lines transfected with wild type or dominant-negative VCP/p97 (WT or H317A, top panel) and time course of treatment with kifunensine (30 μM, bottom panel). Fold change in mean GFP fluorescence was normalised to the levels measured for each reporter at the 3 hr time point of MG132 treatment, and thus a degradation score of 3 is equivalent to the impairment induced by 3 hr MG132 treatment. (d) Target composition of the shRNA library. (e) Overview of the functional genomic screen. (f) Hierarchically clustered heat map of the normalised fold change in mean GFP fluorescence of ERAD reporter lines in response to shRNA-mediated knockdown of ERAD components. The normalisation and colour scale are the same as in panel (c). (g) Functional data from the heat map shown in panel F were mapped onto the refined Hrd1 physical interaction network () to provide an integrated snapshot of substrate-specific functional requirements for Hrd1 network components.

John C. Christianson, et al. Nat Cell Biol. ;14(1):93-105.
8.
Figure 6

Figure 6. From: Defining human ERAD networks through an integrative mapping strategy.

Characterisation of the novel Hrd1-binding partner FAM8A1. (a) Domain structure and interaction network of FAM8A1. (b) Immunoprecipitation with anti-FAM8A1 from HEK293 DIG soluble lysates were analysed by immunoblotting with the indicated antibodies. (c) Consensus TOPCONS prediction of FAM8A1 membrane orientation (http://topcons.cbr.su.se). Reliability index indicates the likelihood for consensus prediction at each position using a sliding 21 amino acid window. (d) HEK293 membrane fractions incubated with 1 M NaCl, 0.1 M Na2CO3 pH 12, or 1% SDS. Following 100,000xg centrifugation, equal volumes of soluble (S) and pellet (P) were analysed by Western blot with anti-FAM8A1. (e) HeLa cells expressing S-FAM8A1 or Hrd1-S were permeabilised with DIG or TX-100 to allow antibody access to cytosolic epitopes or cytosolic and luminal epitopes, respectively, immunostained and analysed by fluorescence microscopy. Scale bar = 10 μm. (f) Hrd1-S expressing HEK293 cell lysates separated on a continuous 10–40% sucrose gradient. S-tagged Hrd1 protein complexes were affinity-purified from each 1 mL fraction (fractions 1–12) or from 150 mg whole cell lysate (10% AP), and analysed by Western blotting for Hrd1 (S-tag), SEL1L, and FAM8A1. (g) Heat map representing the normalised change in mean GFP fluorescence (20,000 cells, n=3) of the indicated ERAD reporter cell lines to transfection with the indicated Hrd1, SEL1L, and FAM8A1 plasmids. DFP indicates dead fluorescent protein, a non-fluorescent GFP variant. Data is represented as a normalised heat map as in .

John C. Christianson, et al. Nat Cell Biol. ;14(1):93-105.

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