1. Global Analysis of Host and Bacterial Ubiquitinome in Response to Salmonella Typhimurium Infection 
Evgenij Fiskin, Tihana Bionda, Ivan Dikic, Christian Behrends 
Molecular Cell 
Volume 62, Issue 6, Pages (June 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 62, 967-981DOI: (10.1016/j.molcel.2016.04.015)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 Host Ubiquitinome upon Salmonella Infection
(A) The diGly proteomics workflow 0.5 and 2 hr pi is shown.
(B) The diGly sites derived from human proteins across 16 experiments (Ex) are shown.
(C) Log2 (H:L) plots for diGly peptides in HCT116 and HeLa cells are shown.
(D) Pearson’s correlation (R) plots for two representative Ex from HCT116 and HeLa cells. Inset shows Pearson’s correlation coefficients (R2).
(E) Venn diagrams of diGly peptides in HCT116 and HeLa cells 0.5 hr pi. The diGly peptide numbers are indicated.
(F) Overlap between diGly sites and their corresponding proteins in both cell lines is shown.
(G and H) Correlation of log2 (H:L) for diGly peptides between cell lines 0.5 (G) and 2 hr (H) pi.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Salmonella-Dependent Ubiquitination Sites
(A) Overlap between up- or downregulated diGly sites and their corresponding proteins in both cell lines 0.5 and 2 hr pi is shown.
(B) Distribution of regulated ubiquitination sites and proteins identified up to four times across four replicate Ex in HCT116 cells 0.5 hr pi is shown.
(C) Annotation enrichment analysis of proteins with regulated diGly sites identified at least twice across eight Ex in HCT116 and/or HeLa cells 0.5 hr pi. The bar graphs show significantly overrepresented pathways (KEGG) as well as gene ontology molecular functions (GOMFs), cellular compartments (GOCCs), and biological processes (GOBPs).
(D) Associations among proteins with regulated diGly sites based on STRING (confidence ≥ 0.7) and BioGrid databases. Selected enrichment categories from (C) are color coded. Node size and color for each protein indicate the number of regulated diGly sites and their mean log2 (H:L) ratio, respectively.
(E–G) Log2 (H:L) ratios for selected regulated diGly sites from two biological replicates in HCT116 (E and G) and in HeLa (F and G) cells.
See also Figure S1.
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5. Figure 3 Early Host Ubiquitinome Dynamics upon Salmonella Infection
(A) The diGly sites quantified 0.5 and 2 hr pi in HCT116 cells are shown.
(B) Heatmap representation of hierarchical clustering of diGly peptides from (A) according to their log2 (H:L) ratios. For each group the number of unique diGly peptides (and their corresponding proteins) is indicated. Data are shown for peptides that were quantified at both time points whereby at least one time point had a log2 (H:L) value ≥1.0 or ≤−1.0.
(C) Log2 (H:L) ratios for selected regulated diGly sites for each group are shown.
(D) Proteomics workflow for wild-type (WT) and ΔSPI-1 Salmonella is shown.
(E) The diGly sites across two Ex from (D) are shown.
(F) Log2 (H:L) ratios for selected regulated diGly sites in biological replicates of ΔSPI-1:WT (D and E) and Control:WT (Figures 1A, 1B, and 2E) are shown.
(G) MG132 proteomics workflow is shown.
(H) Lysates from uninfected or Salmonella (SL1344)-infected HCT116 cells grown in the absence or presence of the proteasome inhibitor MG132 (20 μM) were subjected to SDS-PAGE and immunoblotting.
(I) The diGly sites across two Ex from (G) are shown.
(J) Log2 (H:L) ratios for selected regulated diGly sites from Salmonella-infected HCT116 cells grown in the absence (Figures 1A, 1B, and 2E) or presence (G and I) of MG132 are shown.
(K) K48-Ub proteomics workflow is shown.
(L) Denatured lysates of uninfected or infected (SL1344) HCT116 cells grown in the presence of 20 μM MG132 were subjected to K48-Ub-specific IP followed by SDS-PAGE and immunoblotting.
(M) Proteins quantified by K48-Ub proteomics across two Ex in the presence of MG132 are shown.
(N) Scatterplot of two replicate K48-Ub proteomic Ex (K and M). Selected regulated sites are highlighted in green.
See also Figures S2 and S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 Late Host Ubiquitinome Dynamics upon Salmonella Infection
(A) The diGly proteomics workflow for 6 hr pi is shown.
(B) The diGly sites derived from human proteins across four Ex in HeLa cells are shown.
(C) Log2 (H:L) plots for diGly peptides from (B) are shown.
(D) R plots for two representative Ex. Inset shows R2.
(E) Annotation enrichment analysis of proteins with regulated ubiquitination sites. The bar graph shows significantly overrepresented GOMF, GOCCs, and GOBPs.
(F) Log2 (H:L) ratios for selected regulated diGly sites from (A) are shown.
(G) Log2 (H:L) ratios for selected regulated diGly sites in biological replicates of Control:WT (A–F) and ΔSPI-2:WT (Figures S2F and S2G) are shown.
(H) Proteomics workflow for triple SILAC infection time course is shown.
(I) Heatmap representation of hierarchical diGly peptide clustering according to their log2 (M:L) and (H:M) ratios. For each cluster the number of unique diGly peptides is indicated. Data are shown for those peptides that in at least one condition showed log2 (M:L) or (H:M) values ≥1.0 and ≤−1.0, respectively.
(J) Log2 (H:L) and (H:M) ratios for selected regulated diGly sites for each cluster from (I).
See also Figures S2 and S3.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Ubiquitination of Bacterial Effectors and Outer Membrane Proteins
(A) Heatmap representation of diGly peptide intensities derived from bacterial effectors identified across replicates 0.5 and 2 hr pi in the absence and presence of 20 μM MG132 in HCT116 cells is shown.
(B) Lysates from HCT116 cells infected with WT (SL1344) or SopE-HA-expressing Salmonella in the absence or presence of 20 μM MG132 were subjected to TUBE pull-down followed by SDS-PAGE and immunoblotting.
(C) Immunofluorescence micrographs of WT and TBK1/IKKε DKO MEFs infected with dsRed-expressing Salmonella, fixed 3 hr pi and stained with α-Ub antibody, are shown.
(D) Quantification of Ub-positive Salmonella from (C) is shown.
(E) Heatmap representation of diGly peptide intensities derived from bacterial membrane proteins identified across six replicates in TBK1/IKKε DKO cells 3 hr pi is shown.
(F) Structure and topology of bacterial OMPs from (E) in schematic representation with detected diGly sites marked as red spheres. For bacterial proteins with no available structural information, the structure of close homologs was used. In the case of OmpD, the structure of E. coli OmpA is shown. For FepA, IroN, and FhuE, the structure of E. coli FepA is depicted.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 Role of Infection-Induced CDC42 and LUBAC Ubiquitination
(A) Regulated CDC42 diGly sites upon Salmonella infection are shown.
(B and C) Lysates from HeLa cells expressing HA-CDC42 WT or 3KR (K144R, K183R, and K184R) mutant and infected with Salmonella (SL1344) for 2 hr or left uninfected were subjected to TUBE (B) and PAK1-CRIB (C) pull-down followed by SDS-PAGE and immunoblotting.
(D) Log2 (H:L) ratios of upregulated diGly sites of LUBAC components are shown.
(E) Domain architecture of LUBAC with regulated diGly sites is shown.
(F) Heatmap representation of selected diGly peptide log2 (H:L) ratios ≥1.0 across replicate Ex in HCT116 cells 0.5 and 2 hr pi and treated 15 min with TNF-α is shown.
(G) Lysates from HeLa cells infected with WT (SL1344) or triple effector knockout (ΔSopE, ΔSopE2, and ΔSopB) Salmonella for the indicated time points were subjected to M1-Ub-specific IP followed by SDS-PAGE and immunoblotting.
(H) Lysates from HEK293T cells expressing empty control or GFP-SopE were subjected to M1-Ub-specific IP followed by SDS-PAGE and immunoblotting.
(I) Lysates from HeLa cells infected with Salmonella (SL1344) for the indicated time points in the absence and presence of the indicated concentrations of gliotoxin were subjected to SDS-PAGE and immunoblotting.
(J) Quantification of immunoblots from (I). Data are represented as mean ± SEM (n = 3 independent experiments; ∗p < 0.05, one-tailed paired t test).
(K) Workflow for M1-Ub proteomics is shown.
(L) Denatured lysates of uninfected or SL1344-infected HCT116 cells were subjected to M1-Ub-specific IP followed by SDS-PAGE and immunoblotting.
(M) Human proteins quantified by M1-Ub proteomics from (L). Replicate Ex is shown as a scatterplot. Regulated sites are highlighted in green.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7 Bacterial E3 Ligases and DUBs
(A) Workflow for diGly proteomics comparing cells infected with WT or single bacterial DUB or E3 deletion strains is shown.
(B) The diGly sites in human proteins quantified in two ΔSseL:WT and one ΔSspH2:WT Ex are shown.
(C) Scatterplot of ΔSspH2:WT diGly proteomics experiment. Regulated diGly sites in proteins also identified as enriched in SspH2-IP (D) are highlighted in green, whereas diGly sites in proteins related to the actin cytoskeleton, but not identified in SspH2-IP, are marked in black.
(D) Scatterplot of SILAC-coupled HA-SspH2 IP. Co-enriched proteins are highlighted in green and the bait in red.
(E) Scatterplots of two replicate Ex for ΔSseL:WT. Regulated sites are highlighted in green. Pearson’s correlation coefficient is shown.
(F) Lysates from cells expressing empty vectors, Myc-SseL WT, or C262A were subjected to anti-Myc IP followed by SDS-PAGE and immunoblotting.
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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