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Developmental Studies Hybridoma Bank anti sumo 1 21c7
Antibodies and dilutions used in this study.
Anti Sumo 1 21c7, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "SUMO-mediated regulation of H3K4me3 reader SET-26 controls germline development in C . elegans"

Article Title: SUMO-mediated regulation of H3K4me3 reader SET-26 controls germline development in C . elegans

Journal: PLOS Biology

doi: 10.1371/journal.pbio.3002980

Antibodies and dilutions used in this study.
Figure Legend Snippet: Antibodies and dilutions used in this study.

Techniques Used:



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Developmental Studies Hybridoma Bank anti sumo 1 21c7
Antibodies and dilutions used in this study.
Anti Sumo 1 21c7, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Developmental Studies Hybridoma Bank anti sumo1
Immunoblot analyses of the levels of <t>SUMO1</t> (a) , SUMO2/3 (b) , and GAPDH in the lysates of macrophages infected with S. aureus strains for 24 h post-gentamycin treatment. Using Image Lab software (ChemiDoc), SUMO1 and SUMO2/3 smears were quantified from four different experiments and normalised to GAPDH (lower panels). The fold change chart displays the proportion of SUMOylated proteins recovered from infected cells in comparison to the quantity of SUMOylated proteins in non-infected control macrophages. The data represented are the mean ± SD of four independent biological experiments. **p < 0.01; *p < 0.05; ns, not significant (Kruskal-Wallis test followed by Dunn’s post hoc test).
Anti Sumo1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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A: Endogenously SUMOylated proteins in HL-60 cells. SUMOylated proteins were immunoprecipitated from triple SILAC-labeled HL-60 cells with control (light condition), anti <t>SUMO-1</t> (medium condition) or anti SUMO-2/3 (heavy condition) antibodies and identified by mass spectrometry. Log2-transformed ratios between SUMO-1 or SUMO-2/3 and control IP are represented. Only proteins with more than 2 peptides identified and a Log2 ratio>1 (SUMO-1/control or SUMO-2/control) were selected. The orange dots correspond to selected known SUMO substrates. B: Changes in SUMO-1 and SUMO-2/3 proteomes upon DNR treatment. Scatterplot analysis of SUMO-1 and SUMO-2/3 proteome change (Log2 ratio) in cell treated with DNR (1 µM for 2 hrs) compared to mock-treated cells. Doted lines represent Log2 ratio of -/+ 0.5. Only proteins found to be SUMOylated in A are represented. C: DeSUMOylated proteins are mostly transcriptional regulators . Gene Ontology analysis of the identified down-SUMOylated proteins for SUMO-1 and SUMO-2/3 in response to DNR (log2 ratio <-0.5) were obtained using the Panther Protein Class database . D: The CTCF motif is enriched at SUMO-2/3 binding sites. Motif enrichment search was performed with homer pearl script (findMotifs.pl) on the SUMO-2/3 ChIP-Seq data obtained for mock-treated HL-60. The 3 most enriched motifs are shown. E: SUMO/CTCF overlap on promoter and enhancers. Venn Diagrams for the distribution of the sequences bound by (b) SUMO-2/3, (c) CTCF and (d) both proteins. F: Venn Diagram for the distribution of SUMO-2/3 and CTCF peaks on the whole genome, on promoters (−2 kb;TSS) and putative enhancers in HL-60 cells.
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A: Endogenously SUMOylated proteins in HL-60 cells. SUMOylated proteins were immunoprecipitated from triple SILAC-labeled HL-60 cells with control (light condition), anti <t>SUMO-1</t> (medium condition) or anti SUMO-2/3 (heavy condition) antibodies and identified by mass spectrometry. Log2-transformed ratios between SUMO-1 or SUMO-2/3 and control IP are represented. Only proteins with more than 2 peptides identified and a Log2 ratio>1 (SUMO-1/control or SUMO-2/control) were selected. The orange dots correspond to selected known SUMO substrates. B: Changes in SUMO-1 and SUMO-2/3 proteomes upon DNR treatment. Scatterplot analysis of SUMO-1 and SUMO-2/3 proteome change (Log2 ratio) in cell treated with DNR (1 µM for 2 hrs) compared to mock-treated cells. Doted lines represent Log2 ratio of -/+ 0.5. Only proteins found to be SUMOylated in A are represented. C: DeSUMOylated proteins are mostly transcriptional regulators . Gene Ontology analysis of the identified down-SUMOylated proteins for SUMO-1 and SUMO-2/3 in response to DNR (log2 ratio <-0.5) were obtained using the Panther Protein Class database . D: The CTCF motif is enriched at SUMO-2/3 binding sites. Motif enrichment search was performed with homer pearl script (findMotifs.pl) on the SUMO-2/3 ChIP-Seq data obtained for mock-treated HL-60. The 3 most enriched motifs are shown. E: SUMO/CTCF overlap on promoter and enhancers. Venn Diagrams for the distribution of the sequences bound by (b) SUMO-2/3, (c) CTCF and (d) both proteins. F: Venn Diagram for the distribution of SUMO-2/3 and CTCF peaks on the whole genome, on promoters (−2 kb;TSS) and putative enhancers in HL-60 cells.
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(A) Most sumoylated proteins are associated with chromatin. Fractionation experiments were carried out in normally growing, unmodified yeast and human HeLa cells, then whole cell extracts (WCE) and indicated fractions were analyzed by immunoblot (IB) with indicated antibodies, including antibodies for the yeast Smt3 peptide (“SUMO”), human <t>SUMO1</t> or SUMO2/3 isoforms, and histone H3. (B) Independent duplicate SUMO ChIP-seq experiments were performed in yeast, and sample alignments are shown from Replicate 1, using the Integrative Genomics Viewer (IGV) genomic alignment tool, with reads from the SUMO IP and corresponding inputs over selected short segments of Chromosomes XII and IV. Values correspond to maximum data range (read numbers) for the view shown and blue bars along the bottom represent gene positions, including for two non-RPGs, CCW12 and LYS20 , whose ORF orientations are indicated with arrows. See for a list of yeast genes associated with the 603 SUMO peaks. (C) Pie chart showing fractions of the 603 SUMO ChIP-seq peak set associated with different gene types. Non-RPG refers to protein-coding genes that are not ribosomal protein genes (RPGs). See for detailed description of peak classifications. (D ) Distribution of read counts for the 603 SUMO ChIP-seq peaks, separated by gene type, then ranked by normalized read counts. Normalized read counts were determined using DiffBind tool with two independent ChIP-seq replicates. (E) Sample SUMO peak alignments from Replicate 1 are shown for two RPGs and four non-RPGs. Peak alignments for TBP and Rap1 are also shown, using published ChIP-seq and ChIP-exo datasets, respectively, for comparison with SUMO peak positions (NCBI GEO database accession numbers GSM2870615 and GSE93662, respectively). Values refer to maximum data range (read numbers) for the view shown, and unnormalized alignments were generated using IGV.
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Schematic representation of a quantitative proteome analysis that compares the SUMO proteome of serum‐starved HeLa cells treated for 10 min with or without 100 nM EGF (SILAC labeling and endogenous <t>SUMO1‐</t> and SUMO2/3‐IPs). Scatterplots represent SILAC/SUMO‐IP quantification after EGF treatment from three biological replicates. Each dot represents a protein that is either present in the same ratio between untreated and EGF‐treated cells (dark blue dots) or is significantly more present in one of the samples (red and yellow dots). Left panel: Bar graph depicting mass spectrometry results of 11 proteins with altered SUMOylation (significant hits with P < 0.0001), as well as the three non‐changing proteins SUMO2, RanGAP1, and TRIM28. Right panel: Proteins highlighted in bold in the left panel were validated by SUMO‐IP/immunoblotting from serum‐starved HeLa cells without or with 10‐min EGF stimulation. Time course experiment: Serum‐starved HeLa cells were treated with 100 nM EGF, and samples were harvested at indicated times and subjected to SUMO2 IP followed by immunoblotting with the indicated antibodies. IRF2BP1, IRF2BP2, and TRIM24 are rapidly but transiently deSUMOylated upon EGF treatment.
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Danaher Inc anti sumo 1 21c7 mouse monoclonal antibody55
Schematic representation of a quantitative proteome analysis that compares the SUMO proteome of serum‐starved HeLa cells treated for 10 min with or without 100 nM EGF (SILAC labeling and endogenous <t>SUMO1‐</t> and SUMO2/3‐IPs). Scatterplots represent SILAC/SUMO‐IP quantification after EGF treatment from three biological replicates. Each dot represents a protein that is either present in the same ratio between untreated and EGF‐treated cells (dark blue dots) or is significantly more present in one of the samples (red and yellow dots). Left panel: Bar graph depicting mass spectrometry results of 11 proteins with altered SUMOylation (significant hits with P < 0.0001), as well as the three non‐changing proteins SUMO2, RanGAP1, and TRIM28. Right panel: Proteins highlighted in bold in the left panel were validated by SUMO‐IP/immunoblotting from serum‐starved HeLa cells without or with 10‐min EGF stimulation. Time course experiment: Serum‐starved HeLa cells were treated with 100 nM EGF, and samples were harvested at indicated times and subjected to SUMO2 IP followed by immunoblotting with the indicated antibodies. IRF2BP1, IRF2BP2, and TRIM24 are rapidly but transiently deSUMOylated upon EGF treatment.
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(A) Flowchart followed for UbL signature characterization . Extracts from parental and chemoresistant HL-60 or U937 cells were first supplemented with recombinant UbLs (to avoid rate-limiting amounts of the modifiers) and UbL-vinyl-sulfones (to inhibit UbL deconjugating activities). They were then incubated with protein Protoarrays. After extensive washes, the arrays were incubated with, first, a primary mouse <t>anti-SUMO-1</t> antibody and a rabbit anti-Flag antiserum recognizing the Flag-tag present on the recombinant Ubiquitin added to the reaction and, then, appropriate fluorescent secondary antibodies. Fluorescence signals were processed using the PAA R package. The statistical analysis was performed to identify a UbL signature of chemoresistance, as described in Methods. Three independent experiments were performed for each cell line. ( B) IC 50 of chemosensitive and chemoresistant AML cell lines . IC 50 of chemosensitive parental HL-60 and U937 (wt) cells and of their resistant counterparts (see Materials and Methods) (ARA-R and DNR-R) were assayed after 24 hrs of exposure to drugs. n=3, Mean +/-SEM with * corresponding to p<0.05. (C) Identification of ubiquitylated- and SUMOylated proteins . Normalized Ub and SUMO-1 fluorescence data obtained on all arrays incubated with extracts were compared to averaged signals on control arrays (extracts supplemented with NEM to inhibit UbL conjugation activities) to identify robustly UbL-conjugated proteins. Proteins showing significant differences between the 2 groups when using both the Welch- and the Wilcoxon-Mann-Whitney statistical tests and having mean fluorescence intensities values higher than 800 (arbitrary threshold) on Protoarrays were selected for further analysis. The Venn diagram shows the number or proteins identified as modified by SUMO-1 and/or ubiquitin.
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Image Search Results


Antibodies and dilutions used in this study.

Journal: PLOS Biology

Article Title: SUMO-mediated regulation of H3K4me3 reader SET-26 controls germline development in C . elegans

doi: 10.1371/journal.pbio.3002980

Figure Lengend Snippet: Antibodies and dilutions used in this study.

Article Snippet: anti-SUMO-1 21C7 (1:1,000) , DSHB Cat# SUMO-1 21C7, RRID:AB_2198257.

Techniques:

Immunoblot analyses of the levels of SUMO1 (a) , SUMO2/3 (b) , and GAPDH in the lysates of macrophages infected with S. aureus strains for 24 h post-gentamycin treatment. Using Image Lab software (ChemiDoc), SUMO1 and SUMO2/3 smears were quantified from four different experiments and normalised to GAPDH (lower panels). The fold change chart displays the proportion of SUMOylated proteins recovered from infected cells in comparison to the quantity of SUMOylated proteins in non-infected control macrophages. The data represented are the mean ± SD of four independent biological experiments. **p < 0.01; *p < 0.05; ns, not significant (Kruskal-Wallis test followed by Dunn’s post hoc test).

Journal: bioRxiv

Article Title: The Secreted Tyrosine Phosphatase PtpA promotes Staphylococcus aureus Intramacrophagic Survival Through Decrease of the SUMOylation Host Response

doi: 10.1101/2022.08.31.506036

Figure Lengend Snippet: Immunoblot analyses of the levels of SUMO1 (a) , SUMO2/3 (b) , and GAPDH in the lysates of macrophages infected with S. aureus strains for 24 h post-gentamycin treatment. Using Image Lab software (ChemiDoc), SUMO1 and SUMO2/3 smears were quantified from four different experiments and normalised to GAPDH (lower panels). The fold change chart displays the proportion of SUMOylated proteins recovered from infected cells in comparison to the quantity of SUMOylated proteins in non-infected control macrophages. The data represented are the mean ± SD of four independent biological experiments. **p < 0.01; *p < 0.05; ns, not significant (Kruskal-Wallis test followed by Dunn’s post hoc test).

Article Snippet: Infected macrophages were lysed in 100 μL of 2.5X Laemmli buffer, boiled for 10min at 95°C, sonicated for 10 sec at 50 % amplitude (DIGITAL Sonifier, Model 450-D, BRANSON) and centrifuged for 1min at 12000 x g. Proteins were separated on SDS-PAGEs, transferred to PVDF membranes, and analyzed by Western-blot using an anti-SUMO1 (#21C7, Developmental Studies Hybridoma Bank) or anti-SUMO2/3 antibody (#8A2, Developmental Studies Hybridoma Bank) as primary antibody and a HRP-coupled donkey-anti-mouse antibody as secondary antibody (Jackson ImmunoResearch, Interchim, France).

Techniques: Western Blot, Infection, Software

(a) Intracellular S. aureus strains survival from macrophages overexpressing SUMO1 or SUMO2/3 versus control macrophages (GFP vector). Intracellular bacteria were counted after cell lysis and relative survival is presented as the ratio of intracellular bacteria at 24 h post-gentamicin compared to cells transfected with an empty GFP-vector, considered as 100%. ( b) Macrophages pretreated with ML-792 at 0.5 μM or DMSO were infected with S. aureus strains. Number of intracellular bacteria recovered from macrophages 24 h post-gentamicin was counted and is presented as the ratio of intracellular bacteria compared to cells pretreated with DMSO, considered as 100%. **p < 0.01; *p < 0.05; ns, not significant (Mann-Whitney U test).

Journal: bioRxiv

Article Title: The Secreted Tyrosine Phosphatase PtpA promotes Staphylococcus aureus Intramacrophagic Survival Through Decrease of the SUMOylation Host Response

doi: 10.1101/2022.08.31.506036

Figure Lengend Snippet: (a) Intracellular S. aureus strains survival from macrophages overexpressing SUMO1 or SUMO2/3 versus control macrophages (GFP vector). Intracellular bacteria were counted after cell lysis and relative survival is presented as the ratio of intracellular bacteria at 24 h post-gentamicin compared to cells transfected with an empty GFP-vector, considered as 100%. ( b) Macrophages pretreated with ML-792 at 0.5 μM or DMSO were infected with S. aureus strains. Number of intracellular bacteria recovered from macrophages 24 h post-gentamicin was counted and is presented as the ratio of intracellular bacteria compared to cells pretreated with DMSO, considered as 100%. **p < 0.01; *p < 0.05; ns, not significant (Mann-Whitney U test).

Article Snippet: Infected macrophages were lysed in 100 μL of 2.5X Laemmli buffer, boiled for 10min at 95°C, sonicated for 10 sec at 50 % amplitude (DIGITAL Sonifier, Model 450-D, BRANSON) and centrifuged for 1min at 12000 x g. Proteins were separated on SDS-PAGEs, transferred to PVDF membranes, and analyzed by Western-blot using an anti-SUMO1 (#21C7, Developmental Studies Hybridoma Bank) or anti-SUMO2/3 antibody (#8A2, Developmental Studies Hybridoma Bank) as primary antibody and a HRP-coupled donkey-anti-mouse antibody as secondary antibody (Jackson ImmunoResearch, Interchim, France).

Techniques: Plasmid Preparation, Lysis, Transfection, Infection, MANN-WHITNEY

A: Endogenously SUMOylated proteins in HL-60 cells. SUMOylated proteins were immunoprecipitated from triple SILAC-labeled HL-60 cells with control (light condition), anti SUMO-1 (medium condition) or anti SUMO-2/3 (heavy condition) antibodies and identified by mass spectrometry. Log2-transformed ratios between SUMO-1 or SUMO-2/3 and control IP are represented. Only proteins with more than 2 peptides identified and a Log2 ratio>1 (SUMO-1/control or SUMO-2/control) were selected. The orange dots correspond to selected known SUMO substrates. B: Changes in SUMO-1 and SUMO-2/3 proteomes upon DNR treatment. Scatterplot analysis of SUMO-1 and SUMO-2/3 proteome change (Log2 ratio) in cell treated with DNR (1 µM for 2 hrs) compared to mock-treated cells. Doted lines represent Log2 ratio of -/+ 0.5. Only proteins found to be SUMOylated in A are represented. C: DeSUMOylated proteins are mostly transcriptional regulators . Gene Ontology analysis of the identified down-SUMOylated proteins for SUMO-1 and SUMO-2/3 in response to DNR (log2 ratio <-0.5) were obtained using the Panther Protein Class database . D: The CTCF motif is enriched at SUMO-2/3 binding sites. Motif enrichment search was performed with homer pearl script (findMotifs.pl) on the SUMO-2/3 ChIP-Seq data obtained for mock-treated HL-60. The 3 most enriched motifs are shown. E: SUMO/CTCF overlap on promoter and enhancers. Venn Diagrams for the distribution of the sequences bound by (b) SUMO-2/3, (c) CTCF and (d) both proteins. F: Venn Diagram for the distribution of SUMO-2/3 and CTCF peaks on the whole genome, on promoters (−2 kb;TSS) and putative enhancers in HL-60 cells.

Journal: bioRxiv

Article Title: SUMOylation controls the rapid transcriptional reprogramming induced by anthracyclines in Acute Myeloid Leukemias

doi: 10.1101/2022.04.19.488613

Figure Lengend Snippet: A: Endogenously SUMOylated proteins in HL-60 cells. SUMOylated proteins were immunoprecipitated from triple SILAC-labeled HL-60 cells with control (light condition), anti SUMO-1 (medium condition) or anti SUMO-2/3 (heavy condition) antibodies and identified by mass spectrometry. Log2-transformed ratios between SUMO-1 or SUMO-2/3 and control IP are represented. Only proteins with more than 2 peptides identified and a Log2 ratio>1 (SUMO-1/control or SUMO-2/control) were selected. The orange dots correspond to selected known SUMO substrates. B: Changes in SUMO-1 and SUMO-2/3 proteomes upon DNR treatment. Scatterplot analysis of SUMO-1 and SUMO-2/3 proteome change (Log2 ratio) in cell treated with DNR (1 µM for 2 hrs) compared to mock-treated cells. Doted lines represent Log2 ratio of -/+ 0.5. Only proteins found to be SUMOylated in A are represented. C: DeSUMOylated proteins are mostly transcriptional regulators . Gene Ontology analysis of the identified down-SUMOylated proteins for SUMO-1 and SUMO-2/3 in response to DNR (log2 ratio <-0.5) were obtained using the Panther Protein Class database . D: The CTCF motif is enriched at SUMO-2/3 binding sites. Motif enrichment search was performed with homer pearl script (findMotifs.pl) on the SUMO-2/3 ChIP-Seq data obtained for mock-treated HL-60. The 3 most enriched motifs are shown. E: SUMO/CTCF overlap on promoter and enhancers. Venn Diagrams for the distribution of the sequences bound by (b) SUMO-2/3, (c) CTCF and (d) both proteins. F: Venn Diagram for the distribution of SUMO-2/3 and CTCF peaks on the whole genome, on promoters (−2 kb;TSS) and putative enhancers in HL-60 cells.

Article Snippet: Anti-SUMO-1- (21C7), SUMO2- (8A2) and control- (anti-BrdU, G3G4) hybridomas were obtained from the Developmental Studies Hybridoma Bank (DSHB).

Techniques: Immunoprecipitation, Labeling, Mass Spectrometry, Transformation Assay, Binding Assay, ChIP-sequencing

(A) Most sumoylated proteins are associated with chromatin. Fractionation experiments were carried out in normally growing, unmodified yeast and human HeLa cells, then whole cell extracts (WCE) and indicated fractions were analyzed by immunoblot (IB) with indicated antibodies, including antibodies for the yeast Smt3 peptide (“SUMO”), human SUMO1 or SUMO2/3 isoforms, and histone H3. (B) Independent duplicate SUMO ChIP-seq experiments were performed in yeast, and sample alignments are shown from Replicate 1, using the Integrative Genomics Viewer (IGV) genomic alignment tool, with reads from the SUMO IP and corresponding inputs over selected short segments of Chromosomes XII and IV. Values correspond to maximum data range (read numbers) for the view shown and blue bars along the bottom represent gene positions, including for two non-RPGs, CCW12 and LYS20 , whose ORF orientations are indicated with arrows. See for a list of yeast genes associated with the 603 SUMO peaks. (C) Pie chart showing fractions of the 603 SUMO ChIP-seq peak set associated with different gene types. Non-RPG refers to protein-coding genes that are not ribosomal protein genes (RPGs). See for detailed description of peak classifications. (D ) Distribution of read counts for the 603 SUMO ChIP-seq peaks, separated by gene type, then ranked by normalized read counts. Normalized read counts were determined using DiffBind tool with two independent ChIP-seq replicates. (E) Sample SUMO peak alignments from Replicate 1 are shown for two RPGs and four non-RPGs. Peak alignments for TBP and Rap1 are also shown, using published ChIP-seq and ChIP-exo datasets, respectively, for comparison with SUMO peak positions (NCBI GEO database accession numbers GSM2870615 and GSE93662, respectively). Values refer to maximum data range (read numbers) for the view shown, and unnormalized alignments were generated using IGV.

Journal: PLoS Genetics

Article Title: Dynamic sumoylation of promoter-bound general transcription factors facilitates transcription by RNA polymerase II

doi: 10.1371/journal.pgen.1009828

Figure Lengend Snippet: (A) Most sumoylated proteins are associated with chromatin. Fractionation experiments were carried out in normally growing, unmodified yeast and human HeLa cells, then whole cell extracts (WCE) and indicated fractions were analyzed by immunoblot (IB) with indicated antibodies, including antibodies for the yeast Smt3 peptide (“SUMO”), human SUMO1 or SUMO2/3 isoforms, and histone H3. (B) Independent duplicate SUMO ChIP-seq experiments were performed in yeast, and sample alignments are shown from Replicate 1, using the Integrative Genomics Viewer (IGV) genomic alignment tool, with reads from the SUMO IP and corresponding inputs over selected short segments of Chromosomes XII and IV. Values correspond to maximum data range (read numbers) for the view shown and blue bars along the bottom represent gene positions, including for two non-RPGs, CCW12 and LYS20 , whose ORF orientations are indicated with arrows. See for a list of yeast genes associated with the 603 SUMO peaks. (C) Pie chart showing fractions of the 603 SUMO ChIP-seq peak set associated with different gene types. Non-RPG refers to protein-coding genes that are not ribosomal protein genes (RPGs). See for detailed description of peak classifications. (D ) Distribution of read counts for the 603 SUMO ChIP-seq peaks, separated by gene type, then ranked by normalized read counts. Normalized read counts were determined using DiffBind tool with two independent ChIP-seq replicates. (E) Sample SUMO peak alignments from Replicate 1 are shown for two RPGs and four non-RPGs. Peak alignments for TBP and Rap1 are also shown, using published ChIP-seq and ChIP-exo datasets, respectively, for comparison with SUMO peak positions (NCBI GEO database accession numbers GSM2870615 and GSE93662, respectively). Values refer to maximum data range (read numbers) for the view shown, and unnormalized alignments were generated using IGV.

Article Snippet: Antibodies used for immunoblots are: 1:500 yeast SUMO/Smt3 (y-84; Santa Cruz, sc-28649); 1:3000 GAPDH (Sigma, G9545); histone H3 (1:3000 for yeast immunoblot, 1:20,000 for HeLa immunoblot; Abcam, ab1791); 1:500 SUMO1 (Developmental Studies Hybridoma Bank, 21C7); 1:50 SUMO2/3 (Developmental Studies Hybridoma Bank, 8A2); 1:0000 α-tubulin (Cell Signaling Technology, 2144); 1:1000 HA (Novus, NB600-363); 1:1000 Rpb1 (8WG16; Abcam, ab817); and 1:1000 Rpb3 (Abcam; ab202893).

Techniques: Fractionation, Western Blot, ChIP-sequencing, Generated

Schematic representation of a quantitative proteome analysis that compares the SUMO proteome of serum‐starved HeLa cells treated for 10 min with or without 100 nM EGF (SILAC labeling and endogenous SUMO1‐ and SUMO2/3‐IPs). Scatterplots represent SILAC/SUMO‐IP quantification after EGF treatment from three biological replicates. Each dot represents a protein that is either present in the same ratio between untreated and EGF‐treated cells (dark blue dots) or is significantly more present in one of the samples (red and yellow dots). Left panel: Bar graph depicting mass spectrometry results of 11 proteins with altered SUMOylation (significant hits with P < 0.0001), as well as the three non‐changing proteins SUMO2, RanGAP1, and TRIM28. Right panel: Proteins highlighted in bold in the left panel were validated by SUMO‐IP/immunoblotting from serum‐starved HeLa cells without or with 10‐min EGF stimulation. Time course experiment: Serum‐starved HeLa cells were treated with 100 nM EGF, and samples were harvested at indicated times and subjected to SUMO2 IP followed by immunoblotting with the indicated antibodies. IRF2BP1, IRF2BP2, and TRIM24 are rapidly but transiently deSUMOylated upon EGF treatment.

Journal: EMBO Reports

Article Title: Transient deSUMOylation of IRF2BP proteins controls early transcription in EGFR signaling

doi: 10.15252/embr.201949651

Figure Lengend Snippet: Schematic representation of a quantitative proteome analysis that compares the SUMO proteome of serum‐starved HeLa cells treated for 10 min with or without 100 nM EGF (SILAC labeling and endogenous SUMO1‐ and SUMO2/3‐IPs). Scatterplots represent SILAC/SUMO‐IP quantification after EGF treatment from three biological replicates. Each dot represents a protein that is either present in the same ratio between untreated and EGF‐treated cells (dark blue dots) or is significantly more present in one of the samples (red and yellow dots). Left panel: Bar graph depicting mass spectrometry results of 11 proteins with altered SUMOylation (significant hits with P < 0.0001), as well as the three non‐changing proteins SUMO2, RanGAP1, and TRIM28. Right panel: Proteins highlighted in bold in the left panel were validated by SUMO‐IP/immunoblotting from serum‐starved HeLa cells without or with 10‐min EGF stimulation. Time course experiment: Serum‐starved HeLa cells were treated with 100 nM EGF, and samples were harvested at indicated times and subjected to SUMO2 IP followed by immunoblotting with the indicated antibodies. IRF2BP1, IRF2BP2, and TRIM24 are rapidly but transiently deSUMOylated upon EGF treatment.

Article Snippet: For that we grew hybridoma cells to generate antibodies against SUMO1 (clone 21C7, Developmental Studies Hybridoma Bank) and SUMO2/3 (Clone 8A2, Developmental Studies Hybridoma Bank) and used CELLine bioreactors to harvest antibodies at a concentration of approximately 1 mg/ml.

Techniques: Labeling, Mass Spectrometry, Western Blot

(A) Flowchart followed for UbL signature characterization . Extracts from parental and chemoresistant HL-60 or U937 cells were first supplemented with recombinant UbLs (to avoid rate-limiting amounts of the modifiers) and UbL-vinyl-sulfones (to inhibit UbL deconjugating activities). They were then incubated with protein Protoarrays. After extensive washes, the arrays were incubated with, first, a primary mouse anti-SUMO-1 antibody and a rabbit anti-Flag antiserum recognizing the Flag-tag present on the recombinant Ubiquitin added to the reaction and, then, appropriate fluorescent secondary antibodies. Fluorescence signals were processed using the PAA R package. The statistical analysis was performed to identify a UbL signature of chemoresistance, as described in Methods. Three independent experiments were performed for each cell line. ( B) IC 50 of chemosensitive and chemoresistant AML cell lines . IC 50 of chemosensitive parental HL-60 and U937 (wt) cells and of their resistant counterparts (see Materials and Methods) (ARA-R and DNR-R) were assayed after 24 hrs of exposure to drugs. n=3, Mean +/-SEM with * corresponding to p<0.05. (C) Identification of ubiquitylated- and SUMOylated proteins . Normalized Ub and SUMO-1 fluorescence data obtained on all arrays incubated with extracts were compared to averaged signals on control arrays (extracts supplemented with NEM to inhibit UbL conjugation activities) to identify robustly UbL-conjugated proteins. Proteins showing significant differences between the 2 groups when using both the Welch- and the Wilcoxon-Mann-Whitney statistical tests and having mean fluorescence intensities values higher than 800 (arbitrary threshold) on Protoarrays were selected for further analysis. The Venn diagram shows the number or proteins identified as modified by SUMO-1 and/or ubiquitin.

Journal: bioRxiv

Article Title: Ubiquitin and SUMO conjugation as biomarkers of Acute Myeloid Leukemias response to chemotherapies

doi: 10.1101/825182

Figure Lengend Snippet: (A) Flowchart followed for UbL signature characterization . Extracts from parental and chemoresistant HL-60 or U937 cells were first supplemented with recombinant UbLs (to avoid rate-limiting amounts of the modifiers) and UbL-vinyl-sulfones (to inhibit UbL deconjugating activities). They were then incubated with protein Protoarrays. After extensive washes, the arrays were incubated with, first, a primary mouse anti-SUMO-1 antibody and a rabbit anti-Flag antiserum recognizing the Flag-tag present on the recombinant Ubiquitin added to the reaction and, then, appropriate fluorescent secondary antibodies. Fluorescence signals were processed using the PAA R package. The statistical analysis was performed to identify a UbL signature of chemoresistance, as described in Methods. Three independent experiments were performed for each cell line. ( B) IC 50 of chemosensitive and chemoresistant AML cell lines . IC 50 of chemosensitive parental HL-60 and U937 (wt) cells and of their resistant counterparts (see Materials and Methods) (ARA-R and DNR-R) were assayed after 24 hrs of exposure to drugs. n=3, Mean +/-SEM with * corresponding to p<0.05. (C) Identification of ubiquitylated- and SUMOylated proteins . Normalized Ub and SUMO-1 fluorescence data obtained on all arrays incubated with extracts were compared to averaged signals on control arrays (extracts supplemented with NEM to inhibit UbL conjugation activities) to identify robustly UbL-conjugated proteins. Proteins showing significant differences between the 2 groups when using both the Welch- and the Wilcoxon-Mann-Whitney statistical tests and having mean fluorescence intensities values higher than 800 (arbitrary threshold) on Protoarrays were selected for further analysis. The Venn diagram shows the number or proteins identified as modified by SUMO-1 and/or ubiquitin.

Article Snippet: Next, they were incubated in the washing buffer under agitation (50 rpm) at 4°C for 1 hr with 1 μg/mL of rabbit anti-Flag- (SIGMA, F7425) or mouse anti-SUMO-1 (21C7 from the Developmental Studies Hybridoma Bank) antibodies.

Techniques: Recombinant, Incubation, FLAG-tag, Fluorescence, Conjugation Assay, MANN-WHITNEY, Modification

(A) Identification of UbL-modified biomarkers of AML chemoresistance. Modification levels of the proteins modified by Ubiquitin (left panel) or SUMO-1 (central panel) selected in were compared between all parental (U937 and HL-60) and drug-resistant (ARA-R or DNR-R) sublines. Differentially modified proteins with significant p-values in both Wilcoxon signed-rank- and one sample t-test and with a drug-resistant vs parental cell ratio higher than 1.25 or lower than 0.8 are indicated in red for ubiquitylated proteins and in blue for SUMOylated ones. The Venn diagram shows the overlap between differentially ubiquitylated- and SUMOylated proteins. (B) Identification of UbL-conjugated biomarkers specific for HL-60 and U937 cell resistance to Ara-C or DNR . Statistical analyses between drug-resistant and parental cells were performed separately for U937 and HL-60 cell lines and for each drug resistance. The number of proteins showing a significant p-value in one sample t -test and a ratio between drug-resistant and parental cells higher than 1.5, or lower than 0.66, are shown. (C) Ontology analysis of the UbL signature. An ontology analysis of the 122 proteins of the UbL signature was performed using the Panther software.

Journal: bioRxiv

Article Title: Ubiquitin and SUMO conjugation as biomarkers of Acute Myeloid Leukemias response to chemotherapies

doi: 10.1101/825182

Figure Lengend Snippet: (A) Identification of UbL-modified biomarkers of AML chemoresistance. Modification levels of the proteins modified by Ubiquitin (left panel) or SUMO-1 (central panel) selected in were compared between all parental (U937 and HL-60) and drug-resistant (ARA-R or DNR-R) sublines. Differentially modified proteins with significant p-values in both Wilcoxon signed-rank- and one sample t-test and with a drug-resistant vs parental cell ratio higher than 1.25 or lower than 0.8 are indicated in red for ubiquitylated proteins and in blue for SUMOylated ones. The Venn diagram shows the overlap between differentially ubiquitylated- and SUMOylated proteins. (B) Identification of UbL-conjugated biomarkers specific for HL-60 and U937 cell resistance to Ara-C or DNR . Statistical analyses between drug-resistant and parental cells were performed separately for U937 and HL-60 cell lines and for each drug resistance. The number of proteins showing a significant p-value in one sample t -test and a ratio between drug-resistant and parental cells higher than 1.5, or lower than 0.66, are shown. (C) Ontology analysis of the UbL signature. An ontology analysis of the 122 proteins of the UbL signature was performed using the Panther software.

Article Snippet: Next, they were incubated in the washing buffer under agitation (50 rpm) at 4°C for 1 hr with 1 μg/mL of rabbit anti-Flag- (SIGMA, F7425) or mouse anti-SUMO-1 (21C7 from the Developmental Studies Hybridoma Bank) antibodies.

Techniques: Modification, Software