ddit3 eluates (Novus Biologicals)
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ddit3 eluates
Ddit3 Eluates, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ddit3 eluates/product/Novus Biologicals
Average 94 stars, based on 4 article reviews
Ddit3 Eluates, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ddit3 eluates/product/Novus Biologicals
Average 94 stars, based on 4 article reviews
ddit3 eluates - by Bioz Stars,
2026-05
94/100 stars
Images
1) Product Images from "FET fusion oncoproteins interact with BRD4 and SWI/SNF chromatin remodelling complex subtypes in sarcoma"
Article Title: FET fusion oncoproteins interact with BRD4 and SWI/SNF chromatin remodelling complex subtypes in sarcoma
Journal: Molecular Oncology
doi: 10.1002/1878-0261.13195
Figure Legend Snippet: SWI/SNF subtypes and interactions with FET oncoproteins. (A) Schematic illustration of FET fusion oncoproteins that contain one of the FET proteins (FUS, EWSR1 or TAF15) together with a DNA‐binding transcription‐factor partner. FET oncoproteins are characteristic of many different subtypes of sarcoma and leukaemia, with a few examples listed. Additional FET‐FOPs are continuously being discovered. (B) Schematic illustration of the three SWI/SNF subtypes: cBAF (canonical BAF), PBAF (polybromo BAF) and GBAF/ncBAF (GLTSCR1/L BAF, non‐canonical BAF). Note that some SWI/SNF components are represented by several paralogs, e.g. BAF60A/B/C that are mutually exclusive, and some subunits are unique to one (or two) SWI/SNF subtypes. The reported compositions differ slightly between studies, possibly due to the cell types studied, extraction and purification protocols, and analytical approach. (C) Western blot of 10 µg nuclear extracts (extracted in 500 m m KCl) visualizing SWI/SNF components in myxoid liposarcoma (MLS 402‐91, 2645‐94 and 1765‐92), Ewing sarcoma (EWS TC‐71) and HT1080 fibrosarcoma (wt, EGFP or FUS‐DDIT3‐EGFP) cells using antibodies against core components (BAF155, BAF60A, BAF57 and BAF47), ATPase module components (BRG1, BAF53A and SS18), cBAF (ARID1A and BAF45D), PBAF (PBRM1, ARID2 and BRD7) and GBAF (GLTSCR1, GLTSCR1L and BRD9). Loading controls with all blots are shown in Fig. . (D) Heatmap visualization of gene expression levels for SWI/SNF components in five myxoid liposarcoma tumours (two high‐grade MLS round‐cell type, MLSRC, and three low‐grade MLS) compared to HT1080, from cDNA microarray analysis. In some cases, two or more probes against the same gene were used, shown as separate rows. Gene expression levels are visualized by the normalized log10 ratios in green (less than HT1080) and purple (more than HT1080), and variation in gene expression (standard deviation, SD) is visualized in red. (E) Western blot analysis of DDIT3‐biotin immunoprecipitated (IP) nuclear extracts of MLS 402‐91, visualizing successful co‐IP of the SWI/SNF complex (BRG1, BAF155, BAF57 and BAF47) and the three subtypes: cBAF (ARID1A), PBAF (PBRM1 and BRD7) and GBAF (GLTSCR1L and BRD9) using a direct IP approach. Maximum amount of eluate and around 5% of input was loaded on the gel. All samples were run on the same gel and exposed together. Separating black lines indicate that IgG samples were from a different part of the gel with adjoining lanes not shown. Source data for all WBs (full membranes) are available as supporting information.
Techniques Used: Binding Assay, Extraction, Purification, Western Blot, Gene Expression, Microarray, Standard Deviation, Immunoprecipitation, Co-Immunoprecipitation Assay
Figure Legend Snippet: Composition of FET‐FOP‐bound SWI/SNF complexes.
Techniques Used: Tandem Mass Spectroscopy
Figure Legend Snippet: Sequential salt extraction analysis highlights diverse chromatin binding profiles. (A) Western blot (WB) analysis of MLS 1765‐92, 402‐91 and 2645‐94, and EWS TC‐71 sequential salt extraction (SSE) extracts visualizing FET‐FOPs FUS‐DDIT3 (DDIT3 antibody) or EWSR1‐FLI1 (● FLI1 antibody), as well as EWSR1 and FUS. (B) SSE binding profiles, visualizing the average amount of protein ( n = 3) extracted in the different salt fractions in three MLS cell lines quantified from WB signals in A. FUS‐DDIT3 displayed a distinct binding profile compared to EWSR1 and FUS (adjusted P ‐value 0.0016 and 0.0018, respectively). (C) Western blot analysis of MLS 1765‐92, 402‐91 and 2645‐94, and EWS TC‐71 SSE extracts visualizing SWI/SNF core subunits: BAF170, BAF155, BAF60A, BAF57, BAF47, BRG1, BRM, BAF53A and SS18. (D) SSE binding profiles for SWI/SNF core components ( n = 4), quantified from WB signals in C. Two SWI/SNF core proteins had a slightly different binding profile as illustrated in the two smaller graphs. (E) Western blot analysis of MLS 1765‐92, 402‐91 and 2645‐94, and EWS TC‐71 SSE extracts visualizing SWI/SNF subtype‐specific subunits: ARID1A and ARID1B (cBAF), PBRM1, ARID2 and BRD7 (PBAF), and GLTSCR1, GLTSCR1L, and BRD9 (GBAF). (F) SSE binding profiles for cBAF‐, PBAF‐ and GBAF‐components ( n = 4), quantified from WB signals in E. PBRM1 displayed a distinct binding profile compared to BRD7 ( P = 0.0036). (G) Western blot analysis of MLS 1765‐92, 402‐91 and 2645‐94, and EWS TC‐71 SSE extracts visualizing PRC2‐component EZH2 and control Histone H4. (A, C, E, G) Loading WB: Nuclear SSE extracts (250 m m , 500 m m and 1000 m m ), corresponding to equal initial volume of each salt fraction, were loaded on the gel to directly compare the amount of protein extracted in each fraction. (B, D, F) Binding profiles: Mean +/‐ SD (standard deviation) is shown, n = 3‐4. Statistical significance was determined by 2‐way ANOVA, ** P < 0.01. Detailed plots for each cell line is available in Fig. .
Techniques Used: Extraction, Binding Assay, Western Blot, Control, Standard Deviation
Figure Legend Snippet: Robust interactions between the SWI/SNF complex and FET oncoproteins. (A) Quantitative western blot (QWB) analysis of BRG1‐biotin immunoprecipitated (IP) sequential salt extracts (250, 500 and 1000 m m KCl) in MLS 402‐91 and EWS TC‐71 sarcoma cell lines, visualizing SWI/SNF components (BRG1, ARID1A, BAF155 and BAF57), FET‐FOPs FUS‐DDIT3 and EWSR1‐FLI1 (antibody against C‐terminal partner) and normal FET proteins (EWSR1 and FUS). All IP samples for each cell line were evaluated on the same gel and bands for each antibody were treated equally although separate rectangles are shown for better visualization. Smaller rectangles indicate that the order of the samples were different and cut for visualization purposes. (B) Quantitative western blot analysis of BAF155‐biotin IP nuclear extracts of MLS 402‐91 and 1765‐92 sarcoma cell lines, visualizing SWI/SNF components (BAF155 and BRG1), FET‐FOP FUS‐DDIT3 and normal FET protein EWSR1. Another replicate for MLS 1765‐92 is shown in Fig. . (A‐B) Loading IP‐QWB: Input (I) and eluate (bound, B) samples were diluted relative non‐bound (NB), so that B+NB=100%, as explained in Fig. . Around 5% of input was loaded on the gel.
Techniques Used: Western Blot, Immunoprecipitation
Figure Legend Snippet: BRD4 expression, interactions and inhibition. (A) Schematic visualization of BRD4 long isoform (BRD4‐L) and short (BRD4‐S). Amino acid numbers and potential SUMO post‐translational modification sites (PTM) are indicated. (B) Western blot analysis (WB) of BRD4 isoforms in 10 µg nuclear extracts (extracted in 500 m m KCl) of MLS 402‐91, 2645‐94 and 1765‐92, EWS TC‐71 and HT1080 fibrosarcoma, using a BRD4 antibody (ab128874, N‐term, both isoforms). Analysis with two more BRD4 antibodies (C‐term) are shown in Fig. . (C) Western blot analysis of MLS 1765‐92, 402‐91 and 2645‐94, and EWS TC‐71 SSE extracts visualizing short and long BRD4 isoforms (BRD4 antibody ab128874). Same samples and loading as in Fig. . (D) Quantitative western blot analysis of BRG1‐biotin immunoprecipitated (IP) sequential salt extracts (250, 500 and 1000 m m KCl) in MLS 402‐91 and EWS TC‐71, visualizing BRD4 isoform co‐IP (BRD4 antibody ab128874). Same samples and loading as in Fig. . (E) Western blot analysis of BRG1‐biotin immunoprecipitated nuclear extracts of HT1080 fibrosarcoma cells transiently transfected (24h) with FUS‐DDIT3‐EGFP, EWSR1‐FLI1‐EGFP or EGFP control (R1) visualizing successful co‐IP of the SWI/SNF complex (BAF57) and BRD4. R2‐R3 are displayed in Fig. S4E. (F) Cell viability dose response curves of MLS cell lines 2645‐94, 402‐91 and 1765‐92, EWS TC‐71, HT1080 fibrosarcoma and fibroblasts (F470) after BRD4 inhibition (AZD5153, 72h). Mean +/‐ SD (standard deviation) is shown, n = 12 (2 biological, 6 technical replicates each). (G) Western blot analysis of BRG1‐biotin immunoprecipitated nuclear extracts of MLS 402‐91 control and BRD4‐inhibited cells (BRD4i, AZD5153, 24 h 500 n m ) visualizing SWI/SNF components (BRG1, BAF57 and BAF47), FUS‐DDIT3, normal FET protein EWSR1 and BRD4. (E, G) Loading IP‐WB: Maximum amount of eluate (B) and non‐bound (NB) were loaded on the gel. Input (I) samples were diluted relative NB and around 5% of input was loaded.
Techniques Used: Expressing, Inhibition, Modification, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Control, Standard Deviation
Figure Legend Snippet: ChIP sequencing reveals co‐localization of FUS‐DDIT3, SWI/SNF components and BRD4. (A) ChIP‐seq peak profiles of BAF155, BRG1 and FUS‐DDIT3 in MLS 402‐91 ± 3 kb surrounding TSS (transcription start site). (B) Bar chart showing the genomic distribution of BAF155, BRG1 and FUS‐DDIT3 ChIP‐seq peaks in MLS 402‐91. The majority of peaks are located in promotors close to the TSS, in introns or distal intergenic sites. UTR: mRNA untranslated region. (C) Venn diagram depicting overlap of BAF155, BRG1 and FUS‐DDIT3 ChIP binding sites and annotated genes in MLS 402‐91. The same gene may appear multiple times. The top de novo motif identified with Homer motif discovery for the combined binding sites is shown. (D) Significantly enriched gene sets from the “Reactome” gene set collection using the 4461 unique genes bound by FUS‐DDIT3 and at least one SWI/SNF component. Top 10 based on gene ratio is shown. Gene count is indicated by dot size and P (adjusted)‐value by colour. (E) Venn diagram depicting overlap of genes significantly regulated by ectopic FUS‐DDIT3‐EGFP expression (adjusted P ‐value ≤ 0.05 and Log2 fold change≥ 1) and unique genes bound by FUS‐DDIT3 and at least one SWI/SNF component. Significant enrichment was determined by Fisher’s exact test ( P < 1e‐10). (F) Venn diagram depicting overlap of BAF155, BRG1 and FUS‐DDIT3 ChIP binding sites in MLS 402‐91 from our dataset combined with BRD4 binding sites from Chen et al. dataset.
Techniques Used: ChIP-sequencing, Binding Assay, Expressing
Figure Legend Snippet: The interactomes of FET oncoproteins are enriched in phase separation propensity and transcriptional components. (A) Significantly enriched Panther protein class for FUS‐DDIT3‐interacting proteins. Percentage of proteins in protein class versus total of proteins matched to a protein class. (B) Significantly enriched gene sets from the “Reactome” and “Gene ontology (GO) biological processes” gene set collections for FUS‐DDIT3‐interacting proteins. Top 10 based on gene ratio is shown. Gene count is indicated by dot size and q‐value by colour. (C) Pie charts of FUS‐DDIT3 and EWSR1‐FLI1‐interacting proteins with phase separation propensity score (PScore) above or below the cutoff at 4. (D) Visualization of phase separation propensity score (PScore) of FET oncoproteins and their parental proteins. Pie chart shows proteins above or below the cutoff at 4. Blue dot indicates proteins with a high phase separation propensity, above the cutoff, visualized by black line. (E) Visualization of phase separation propensity score (PScore) of SWI/SNF components. Pie chart shows proteins above or below the cutoff at 4. Red dot indicates proteins with a high phase separation propensity, above the cutoff, visualized by black line. (F) Schematic visualization of potential BRD4, mediator, RNA polymerase II and FET‐FOP‐bound SWI/SNF complex interactions near chromatin. (G) Immunofluorescence staining of HT1080 cells transiently transfected with FUS‐DDIT3‐EGFP, probed with BRG1/BRD4, BRG1/MED1 and FUS‐DDIT3‐EGFP/BRD4 and analyzed with laser scanning microscopy. Representative images are shown. Scale bar: 5 µm. (H) Pearson’s correlation coefficient for co‐localization of BRG1 vs. BRD4, BRG1 vs. MED1, FUS‐DDIT3 vs. BRD4, FUS‐DDIT3 vs. BRG1 and FUS‐DDIT3 vs. MED1 in HT1080 cells transiently transfected with FUS‐DDIT3‐EGFP. (I) Manders’ split coefficients for co‐localization of BRG1 vs. BRD4, BRG1 vs. MED1, FUS‐DDIT3 vs. BRD4, FUS‐DDIT3 vs. BRG1 and FUS‐DDIT3 vs. MED1 in HT1080 cells transiently transfected with FUS‐DDIT3‐EGFP. The dark grey bar corresponds to the fraction of protein 1 (e.g. BRG1) overlapping with protein 2 (e.g. BRD4) and the light grey corresponds to the fraction of protein 2 (e.g. BRD4) overlapping with protein 1 (e.g. BRG1).
Techniques Used: Immunofluorescence, Staining, Transfection, Laser-Scanning Microscopy