Nature Structural & Molecular Biology: doi: /nsmb.3218
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2 Supplementary Figure 1 Endogenous EGFR trafficking and responses depend on biased ligands. (a) Lysates from HeLa cells stimulated for 2 min. with increasing concentration of ligands were immunoblotted as indicated. (b) Object-based co-localization (see Online Methods) of the experiment shown in Fig. 1d. Values in the graph represent the median ± SD of four images from independent experiments. *, p value<0.02 (Wilcoxon test with alpha 0.05). These results were consistent with those shown in Fig. 1c. (c) Quantification (see Online Methods) of the presence of EGFR in endocytic markers-positive regions upon EGF- or TGF- - stimulation. Values in the graph represent the means ± SD. of three experiments. A.U., arbitrary units.*, p value<0.01. (d) Representative images from (c), showing the presence of EGF- or TGF- -stimulated EGFR (red) in intracellular markers (green)-positive regions at different time intervals. Activated EGFR is in blue. Bar, 5 m. See also Supplementary Data Set 1 for uncropped gels.
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4 Supplementary Figure 2 Assessment of the multilayered proteomics dataset shows overall data quality. (a) Experimental design of MS-based quantitative analysis of EGF- and TGF- -induced signaling dynamics and changes in protein abundance. (b) The correlation of each of the five data sets shows good reproducibility between biological replicates. The distribution of peptide mass error (c) and peptide intensity (d) shows that most of the peptides were identified with high mass accuracy (mass error less than 2 p.p.m.) and high intensity. (e) Number of identified sites and proteins at each time point. See Fig.3a for number of regulated sites at each time point. (f) Distribution of Serine (Ser), Threonine (Thr) and Tyrosine (Tyr) phosphorylated sites identified in this study. (g) Distribution of phosphopeptides with one, two, or more phosphorylated sites. (h) Distribution of ubiquitylated peptides with one, two, or more ubiquitylated sites.
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6 Supplementary Figure 3 Clustering and S-score analyses reveal ligand-dependent regulation of phosphorylation and ubiquitination. (a) Left, clustering based on fuzzy c-means of the 330 phosphorylation sites and 52 ubiquitylated sites quantified at all time points. Right, number of sites in each cluster. Each cluster included both phosphorylated and ubiquitylated sites whose SILAC ratios were as similar as possible within the same cluster and as dissimilar as possible between clusters (see Online Methods). However, sites with similar patterns were not necessarily co-regulated and sites could belong to more than one cluster. For instance, phosphorylated tyrosine 998 on EGFR belonged to cluster 4 in case of EGF-stimulated cells and to cluster 1 in case of TGF- -treated samples. This indicates a different regulation based on experimental conditions. Ubiquitylated lysine K867 on EGFR belonged to cluster 4 in case of EGF-stimulated cells, showing similar regulation as tyrosine 998. On the contrary, it was found in cluster 5 in TGF- -treated samples, suggesting again context-dependent regulation. (b) GO terms enrichment analysis of clustering shown in (a). (c) Phosphorylation and ubiquitylation sites were portioned in five equally sized groups based on S-score (Rigbolt, K.T. et al. Sci Signal 4, rs3, 2011), and each group was tested for over-represented GO Biological Process terms compared to the sites from the remaining groups. Enriched GO terms for the most stimulus-specific sites are found at the left (S score<40) whereas events common to both stimuli increase towards the right (S score >40). The results are color-coded to indicate the S-score percentile bins where the GO term was found over-represented. GO terms with S- score <40, whose members are shown in (d), are highlighted in the magenta box. (d) Overview of the 66 ubiquitylated proteins that are differentially regulated between EGF and TGF- color-coded based on the clustering shown in (a). Two proteins have been found to be specifically regulated by TGF- the late proteome analysis (magenta font). in
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8 Supplementary Figure 4 The cross-talk between phosphorylation and ubiquitination is stimulus dependent. Heat maps of the 25 doubly modified proteins shown in Fig.4d-e and color-coded according to regulation upon EGF or TGF- Phosphorylated proteins are shown on the left and ubiquitylated proteins on the right. The validated candidate Rab7A is shown in magenta.
9 Supplementary Figure 5 RAB7 phosphorylation on Y183 is enhanced by EGF. (a) Experimental design of MS-based quantitative analysis of EGF- or TGF- -dependent phosphoproteome in HeLa cells stimulated for 1, 8, 90 min.. (b) The correlation between EGF vs. TGF- -stimulated cells in two biological replicates shows an overall similar phosphoproteome. The proteins whose phosphorylated site is differentially regulated upon one or the other stimulus are highlighted in colors (blue, EGF regulated; green, TGF- regulated). (c, d) Quantitation by MS of Erk1 and Erk2 Y202/T204-containing doubly phosphorylated peptide (c) and Rab7 Y183 phosphorylated peptide (d) upon EGF (blue) or TGF- (green) stimulation. Values are the median ± SD of two replicates. A.U., arbitrary units In (d) the values of the experiment shown in Supplementary Table 2 are also reported for comparison.. Data presented in (c) confirmed the temporal profiles of Erk activation shown in Fig. 2d-f and in Supplementary Tables 2 and 6.
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11 Supplementary Figure 6 RAB7 phosphorylation on Y183 influences the RAB7 interactome. (a) Experimental design of MS-based quantitative analysis of EGF-stimulated cells transfected with Rab7 or its mutant. (b) The correlation of the two biological replicates shows good reproducibility. (c) Ion extracted chromatogram of the wild type and Y F mutant Rab7 peptide. (d) Full-scan MS (left) and representative MS/MS spectrum (right) of the ubiquitylated K191 peptide of wt and mutant Rab7. (d) Table with information of the chemical composition of the Rab7 peptides. (e) List of the significant interactors of mutant Rab7 with a role in endocytosis upon EGF stimulation. (f) Lysates from notstimulated HeLa cells transfected with Rab7-GFP were immunoblotted as indicated. See also Supplementary Data Set 1 for uncropped gels.
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13 Supplementary Figure 7 The phosphorylation and ubiquitination patterns on EGFR and EGFR interactors are ligand dependent. (a, b) Lysates from stimulated cells were immunoprecipitated and immunoblotted with the indicated antibodies. py, anti-phosphorylated Tyr antibody. py1045, anti-phosphorylated Tyr1045 antibody. This antibody recognizes Y1069 on EGFR sequence. py1068, anti-phosphorylated Tyr1068 antibody. This antibody recognizes Y1092 on EGFR sequence. P4D1, antibody recognizing both polyubiquitin and monoubiquitin (Haglund, K. et al., Nat Cell Biol 5, ). FK1, antibody recognizing only polyubiquitin (both as ubiquitin monomers and polymers) but not single ubiquitin monomers 2. Since EGFR was immunoreactive with P4D1 but not with FK1 antibody, we concluded that the receptor was monoubiquitinated. Monoubiquitination has a role in lysosomal degradation, protein-protein interaction or recruitment to a certain sub-cellular compartment (Komander, D. & Rape, M. Annu Rev Biochem 81, ). K48, antibody recognizing ubiquitin linked to Lysine 48, a marker for proteasomal degradation or transcription factor regulation Komander, D. & Rape, M. Annu Rev Biochem 81, ). K63, antibody recognizing ubiquitin linked to Lysine 63, a marker for lysosomal degradation, signaling, receptor trafficking or protein-protein interaction 3. (c) Lysates from stimulated HeLa cells were immunoblotted as indicated before immunoprecipitation. (d) Summary of the estimated occupancy of phosphorylated tyrosine and ubiquitylated lysine residues on EGFR at different time points. (e) Top, lysates from stimulated HeLa cells were immunoblotted as indicated before immunoprecipitation of HRS (also known as HGS). Bottom, log2 ratio of the difference between EGF and TGF- of the identified phosphorylated tyrosine residues on HRS at 8 min. stimulation These data together indicate that HRS was more phosphorylated on tyrosine residues upon EGF than upon TGFstimulation. (f) Overview of the PTM regulation on the EGFR interactors identified by MS. Each square indicates a time point and the color-scale represents the difference between the regulation upon EGF divided by the regulation upon TGF- stimulation. (g, h) Lysates from stimulated HeLa cells were immunoprecipitated and immunoblotted with the indicated antibodies. See also Supplementary Data Set 1 for uncropped gels.
Department of Chemistry, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, H3C 3J7, Canada.
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www.sciencesignaling.org/cgi/content/full/7/322/ra38/dc1 Supplementary Materials for Dynamic Reprogramming of Signaling Upon Met Inhibition Reveals a Mechanism of Drug Resistance in Gastric Cancer Andrea
PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland
AD Award Number: W81XWH-12-1-0248 TITLE: Targeted Inhibition of Tyrosine Kinase-Mediated Epigenetic Alterations to Prevent Resurgence of Castration-Resistant Prostate Cancer PRINCIPAL INVESTIGATOR: Dr.
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DOI: 10.1038/ncb2566 Figure S1 CDKL5 protein expression pattern and localization in mouse brain. (a) Multiple-tissue western blot from a postnatal day (P) 21 mouse probed with an antibody against CDKL5.
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/8/385/ra70/dc1 Supplementary Materials for The interaction of heparan sulfate proteoglycans with endothelial transglutaminase-2 limits VEGF 165 -induced angiogenesis
Proteins: Proteomics & Protein-Protein Interactions Part I
Proteins: Proteomics & Protein-Protein Interactions Part I Jesse Rinehart, PhD Department of Cellular & Molecular Physiology Systems Biology Institute DNA RNA PROTEIN DNA RNA PROTEIN Proteins: Proteomics
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SUPPLEMENTARY INFORMATION doi:10.1038/nature13418 Supplementary Results: USP30 opposes autophagic flux In HEK-293 cells, USP30 overexpression increased basal LC3-II levels, dependent on enzymatic activity,
Supplemental Figure 1
Supplemental Figure 1 1a 1c PD-1 MFI fold change 6 5 4 3 2 1 IL-1α IL-2 IL-4 IL-6 IL-1 IL-12 IL-13 IL-15 IL-17 IL-18 IL-21 IL-23 IFN-α Mut Human PD-1 promoter SBE-D 5 -GTCTG- -1.2kb SBE-P -CAGAC- -1.kb
Nature Genetics: doi: /ng Supplementary Figure 1. SEER data for male and female cancer incidence from
Supplementary Figure 1 SEER data for male and female cancer incidence from 1975 2013. (a,b) Incidence rates of oral cavity and pharynx cancer (a) and leukemia (b) are plotted, grouped by males (blue),
Supplementary Figure 1. Spatial distribution of LRP5 and β-catenin in intact cardiomyocytes. (a) and (b) Immunofluorescence staining of endogenous
Supplementary Figure 1. Spatial distribution of LRP5 and β-catenin in intact cardiomyocytes. (a) and (b) Immunofluorescence staining of endogenous LRP5 in intact adult mouse ventricular myocytes (AMVMs)
Nature Immunology doi: /ni.3268
Supplementary Figure 1 Loss of Mst1 and Mst2 increases susceptibility to bacterial sepsis. (a) H&E staining of colon and kidney sections from wild type and Mst1 -/- Mst2 fl/fl Vav-Cre mice. Scale bar,
Proteins are sometimes only produced in one cell type or cell compartment (brain has 15,000 expressed proteins, gut has 2,000).
Lecture 2: Principles of Protein Structure: Amino Acids Why study proteins? Proteins underpin every aspect of biological activity and therefore are targets for drug design and medicinal therapy, and in
genome edited transient transfection, CMV promoter
Supplementary Figure 1. In the absence of new protein translation, overexpressed caveolin-1-gfp is degraded faster than caveolin-1-gfp expressed from the endogenous caveolin 1 locus % loss of total caveolin-1-gfp
Supplementary Figure 1 (previous page). EM analysis of full-length GCGR. (a) Exemplary tilt pair images of the GCGR mab23 complex acquired for Random
S1 Supplementary Figure 1 (previous page). EM analysis of full-length GCGR. (a) Exemplary tilt pair images of the GCGR mab23 complex acquired for Random Conical Tilt (RCT) reconstruction (left: -50,right:
Supplementary Table 1. Properties of lysates of E. coli strains expressing CcLpxI point mutants
Supplementary Table 1. Properties of lysates of E. coli strains expressing CcLpxI point mutants Species UDP-2,3- diacylglucosamine hydrolase specific activity (nmol min -1 mg -1 ) Fold vectorcontrol specific
TITLE: Effect of MUC1 Expression on EGFR Endocytosis and Degradation in Human Breast Cancer Cell Lines
AD AWARD NUMBER: W81XWH-06-1-0464 TITLE: Effect of MUC1 Expression on EGFR Endocytosis and Degradation in Human Breast Cancer Cell Lines PRINCIPAL INVESTIGATOR: Rachid M El Bejjani CONTRACTING ORGANIZATION:
7SK ChIRP-seq is specifically RNA dependent and conserved between mice and humans.
Supplementary Figure 1 7SK ChIRP-seq is specifically RNA dependent and conserved between mice and humans. Regions targeted by the Even and Odd ChIRP probes mapped to a secondary structure model 56 of the
Supplementary Table 1. Metabolic parameters in GFP and OGT-treated mice
Supplementary Table 1. Metabolic parameters in GFP and OGT-treated mice Fasted Refed GFP OGT GFP OGT Liver G6P (mmol/g) 0.03±0.01 0.04±0.02 0.60±0.04 0.42±0.10 A TGs (mg/g of liver) 20.08±5.17 16.29±0.8