Journal: bioRxiv

Article Title: The tyrosine kinase inhibitor GNF-7 targets senescent cells through allosteric activation of GCN2

doi: 10.1101/2025.08.29.673062

Figure Lengend Snippet: ( A ) Dot plot representing significantly upregulated biological processes in senescent HT-1080 T cells upon treatment with GNF-7 (200nM, 6 h). The X-axis indicates the enrichment score (-Log10pvalue). For the analysis the top 100 hits sorted by False Discovery Rate (FDR) and with a positive log2FoldChange were selected. Senescence was induced by exposure to doxycycline (1μg/ml) for 6 days. ( B ) WB analysis of phosphorylated GCN2 (pGCN2) and CHOP levels in proliferating and senescent HT’1080 cells. GNF-7 was used at the indicated concentrations for 3 h. Senescence was induced by palbociclib (5 μM) for 7 days. ( C ) Heatmap representing the EGFP median fluorescence intensity in DLD-1-CHOP EGFP cells after treatment with the indicated drugs for 24 h. Cells were infected with mCherry-expressing lentiviral vectors carrying sgRNAs to target the four ISR kinases. Control cells were infected with the empty vector. EGFP MFI ratios between mCherry-positive (KO) and negative (WT) cells were calculated and normalized to the control levels for each treatment: untreated, Tigecycline 10 μM, Neratinib 500 nM and GNF-7 200 nM. ( D ) Competition assay in wild type (red, WT) and GCN2-deficient (green, GCN2 KO ) HT-1080 cells cultured for 12 days in the presence or absence of 50 nM GNF-7. (E) Percentage of apoptotic cells (measured by FACS as Annexin V positive and PI negative) upon GNF-7 treatment in proliferating cells, as well as senescent WT and GCN2 KO HT-1080 cells. Senescence was induced by palbociclib (5 μM) for 7 days. GNF-7 was used at the indicated doses, and navitoclax at 5 μM.

Article Snippet: To monitor CHOP levels, we used the lentiviral plasmid pCLX-CHOP-dGFP (Addgene, 71299).

Techniques: Fluorescence, Infection, Expressing, Control, Plasmid Preparation, Competitive Binding Assay, Cell Culture

Journal: Biomedicines

Article Title: Protective Effects of Trimetazidine and Dexmedetomidine on Liver Injury in a Mesenteric Artery Ischemia–Reperfusion Rat Model via Endoplasmic Reticulum Stress

doi: 10.3390/biomedicines12102299

Figure Lengend Snippet: Representative light microscopic screen images of liver tissue sections incubated with CHOP primary antibody. ( A ) (×20). Control group: In the liver tissue sections from the control group, hepatocytes with a normal structure (arrow) were observed to be CHOP-negative (CHOP positivity score: 0(0-0)). ( B ) (×20). I/R group: In the intralobular areas, primarily in Zone 1, hepatocytes showing intense CHOP positivity (spiral arrow) are observed (CHOP positivity score: 2(1-2)). ( C ) (×20). I/R+TMZ group: A decrease in hepatocytes showing CHOP positivity was observed in the perinobular areas, particularly in the intralobular regions (CHOP positivity score: 1(0-1)). ( D ) (×20). I/R+DEX group: A decrease in hepatocytes showing intense immunopositivity in the intralobular and perilobular areas was observed, with a widespread presence of CHOP-negative hepatocytes (arrow, CHOP positivity score: 0.5 (0-1)).

Article Snippet: The liver tissue slices were analyzed via immunohistochemistry labeling with primary antibodies against CHOP (E-AB-70087, 1/300, Elabscience, Houston, TX, USA), GPR78 (SC-13539, 1/350, Santa Cruz Biotechnology Inc. Dallas, TX, USA), 8-hydroxy-2′-deoxyguanosine (8-OHdG) (Santa Cruz, SC-66036, 1/200, Santa Cruz Biotechnology Inc., Dallas, TX, USA), cleaved caspase-3 (Abcam, ab4051, 1/100), anti-Bax ab32503, 1/200, (Abcam, Cambridge, UK), and anti-Bcl-2 (Abcam, ab32124, 1/200).

Techniques: Incubation, Control

Journal: Biomedicines

Article Title: Idebenone Protects against Spontaneous Chronic Murine Colitis by Alleviating Endoplasmic Reticulum Stress and Inflammatory Response

doi: 10.3390/biomedicines8100384

Figure Lengend Snippet: Effect of idebenone on mRNA levels of endoplasmic reticulum stress (ER stress) markers in the Winnie model of spontaneous chronic colitis. Gene expression of ( A ) GRP78 , ( B ) CHOP , ( C ) ATF6 , ( D ) XBP-1 and ( E ) PERK were determined for the proximal (PC) and distal colon (DC) for all the three groups. mRNA levels were normalized to GAPDH gene and presented as fold change. Data represents mean ± SEM ( n = 4/group) using One-way ANOVA followed by Tukey’s post-test, where * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: The membranes were then blocked in 5% skim milk prepared in TBST and incubated overnight with primary antibody against NQO-1 (1:1000, ab34173, Abcam), GRP78 (1:1000, NBP1-06274, Novus biologicals) CHOP (1:1000, NBP2-13172, Novus Biologicals) and β-actin (1:8000, NB600-503, Novus Biologicals) at 4 °C.

Techniques: Gene Expression

Journal: Biomedicines

Article Title: Idebenone Protects against Spontaneous Chronic Murine Colitis by Alleviating Endoplasmic Reticulum Stress and Inflammatory Response

doi: 10.3390/biomedicines8100384

Figure Lengend Snippet: Effect of idebenone on endoplasmic reticulum stress (ER stress) and oxidative stress in spontaneous chronic colitis in Winnie mice. ( A ) protein levels of GRP78, CHOP and NQO-1 were analyzed using western blotting of tissue samples from the distal colon (DC), ( B – D ) densitometry of GRP78, CHOP and NQO-1 expression. Band densities were normalized to β-actin. ( E ) malondialdehyde (MDA) levels in distal colon. Data represents mean ± SEM ( n = 3/group). Statistical significance was determined using One-way ANOVA followed by Tukey’s post-test, where * p < 0.05, ** p < 0.01 and *** p < 0.001 and ns: nonsignificant.

Article Snippet: The membranes were then blocked in 5% skim milk prepared in TBST and incubated overnight with primary antibody against NQO-1 (1:1000, ab34173, Abcam), GRP78 (1:1000, NBP1-06274, Novus biologicals) CHOP (1:1000, NBP2-13172, Novus Biologicals) and β-actin (1:8000, NB600-503, Novus Biologicals) at 4 °C.

Techniques: Western Blot, Expressing

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: The three normal RNA‐binding FET proteins consist of N‐terminal repetitive and structurally disordered domains (NTD: N‐terminal domain), central RNA‐binding domains (RRM: RNA recognition motifs), zinc finger domains (ZF), and degenerated repeat regions (RGG: RGG repeat regions). The GST‐tagged FET‐NTD baits shown were used for pulldown experiments and represent the shortest parts commonly present in the FET fusion oncoproteins. The FET‐binding motif is a conserved sequence required for complex formation between the three FET proteins. Amino acid numbers are indicated. Schematic illustration of a representative FET fusion protein (FUS‐DDIT3 type II) and its parental proteins. DBD: DNA‐binding domain, LZ: leucine zipper domain. Type I and type II show locations of the two most common MLS fusion breakpoints in FUS. Coomassie staining of SDS–PAGE‐separated pulldown samples with/without cell extracts (C.E) using GST‐tagged FET‐NTDs as baits. Sepharose with bound GST was included as control. Background from recombinant protein baits (shown by white arrowheads) as well as smaller partial recombinant products is visualized in the left panel (C.E −). Several high‐molecular‐weight proteins are retained by the FET‐NTDs (C.E +). Black arrowheads indicate protein bands and gel parts analyzed by mass spectrometry (see also Table EV1 ). Immunoblot analysis (IB) of pulldown samples with FET‐NTD baits with/without cell extracts (C.E). Antibodies against ARID1A, BRG1, and BAF60A were used for detection of SWI/SNF components. Input samples diluted 1:5 were included as a control. In situ proximity ligation assays (PLA) using antibodies against BRG1, ARID1A, DDIT3, and C‐terminal parts of normal FUS show protein complexes containing FUS/BRG1, FUS/ARID1A, FUS‐DDIT3/BRG1, and FUS‐DDIT3/ARID1A as red fluorescent spots in nuclei of MLS cell lines 2645‐94 and 402‐91. C‐terminal parts of FUS are not present in the FUS‐DDIT3 fusion protein, and normal DDIT3 is not expressed in these cell lines. Merged images also include DAPI nuclear counterstain in blue. Combinations of primary antibodies are used to detect interactions (left panel). In control experiments (right panel), one primary antibody is omitted to evaluate the background fluorescent signals. Scale bars = 10 μm. Source data are available online for this figure.

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: RNA Binding Assay, Binding Assay, Sequencing, Staining, SDS Page, Control, Recombinant, High Molecular Weight, Mass Spectrometry, Western Blot, In Situ, Ligation

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: Summary of SWI/SNF components as well as FET proteins (wild‐type and fusion oncoproteins) in the bound fraction after BRG1 Co‐IP of MLS 402‐91, MLS 2645‐94, EWS TC‐71, and EWS IOR/CAR, after DDIT3 Co‐IP of MLS 402‐91 and after FLI1 Co‐IP of EWS TC‐71 as verified by mass spectrometry (MS), and by immunoblot (IB) for key proteins. Note that some SWI/SNF components are represented by one of several alternative variants, for example BAF45A‐D. Lists of peptides for each mass spectrometry hit are shown in Tables EV2 and EV3

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: Mass Spectrometry, Western Blot, Tandem Mass Spectroscopy

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: Immunoblot analysis (IB) of proteins co‐immunoprecipitated with BRG1. Detection with antibodies against BRG1, EWSR1, and FUS‐DDIT3 in MLS cell lines 402‐91 and 2645‐94. Immunoblot analysis (IB) of proteins co‐immunoprecipitated with BRG1. Detection with antibodies against and BRG1 and EWSR1 in EWS cell lines TC‐71 and IOR/CAR. CE: cytoplasmic extract, NE: nuclear extract, B FA : bound proteins eluted with 1% formic acid, B LDS : bound proteins eluted with LDS sample buffer, NB: non‐bound proteins, not captured by the antibody. Source data are available online for this figure.

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: Western Blot, Immunoprecipitation

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: Immunoblot analysis (IB) of proteins co‐immunoprecipitated with nuclear extracted SWI/SNF from MLS cell line 402‐91 and EWS cell line TC‐71. Antibodies against BRG1, DDIT3 (FUS‐DDIT3), the C‐terminal parts of normal FUS and EWSR1, and FLI1 (EWSR1‐FLI1) were used for detection. In order to directly quantify the fraction of bound and non‐bound protein, relative amounts of protein for each IP‐sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. One representative immunoblot is shown. Immunoblots from all replicates are shown in the source data. Graphs showing the percentage of bound to total (bound + non‐bound) signal intensities from immunoblots for FUS‐DDIT3, EWSR1, and FUS in MLS 402‐91 cell line and EWSR1‐FLI1, EWSR1, and FUS in EWS TC‐71 cell line. Mean ± SEM is shown with individual replicates indicated by circles ( n = 3). Student's t ‐test, * P < 0.05, ** P < 0.01, *** P < 0.001. Original data for all quantifications, including P ‐values, are shown as source data. Immunoblot analysis of proteins co‐immunoprecipitated with nuclear extracted SWI/SNF from the model cell line HT1080 with overexpressed FUS‐DDIT3‐dsRED, EWSR1‐FLI1‐EGFP, or dsRED is shown as representative immunoblots. Antibodies against BRG1, DDIT3 (FUS‐DDIT3‐dsRED), the C‐terminal parts of normal FUS and EWSR1, EZH2, and FLI1 (EWSR1‐FLI1‐EGFP) were used for detection. In order to directly quantify the fraction of bound and non‐bound protein, relative amounts of protein for each IP‐sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. Immunoblots from all replicates are shown in the source data (with either dsRED‐ or EGFP‐tagged proteins expressed). Graphs showing the percentage of bound to total (bound + non‐bound) signal intensities from immunoblots for the fusion protein (FUS‐DDIT3 or EWSR1‐FLI1), EWSR1, FUS, and EZH2 in the model cell line HT1080 with overexpressed FUS‐DDIT3‐dsRED/‐EGFP ( n = 4, circles), EWSR1‐FLI1‐EGFP ( n = 3, squares) or dsRED/EGFP ( n = 4, triangles). Mean ± SEM is shown with individual replicates indicated. Student's t ‐test, no significant changes, P > 0.05. Original data for all quantifications, including P ‐values, are shown as source data. Four alternative models for binding of normal and oncogenic FET proteins to SWI/SNF. (I): FET oncoproteins bind directly to SWI/SNF. (II): normal FET proteins form homo‐ and hetero‐complexes and mediate binding of FET oncoproteins. (III): FET oncoproteins and normal FET proteins bind to different variants of the SWI/SNF complex with distinct biochemical compositions. (IV): FET oncoproteins and normal FET proteins bind to different sites on the SWI/SNF complex. Source data are available online for this figure.

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: Western Blot, Immunoprecipitation, Binding Assay

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: Immunoblot analysis (IB) of histone modifications in stably transfected HT1080 cell lines expressing EGFP, FUS‐DDIT3‐EGFP, or EWSR1‐FLI1‐EGFP. Antibodies against H3K27Ac, H3K27me3, H3K4me3, and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount. Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion oncoproteins and EGFP. One representative immunoblot is shown. More immunoblots including a second stable biological replicate and analysis of histone modifications after transient transfection (24 and 48 h) are shown in Fig A–D. Graphs showing amount of protein (H3K27me3, H3K4me3, ratio H3K27me3/H3K4me3, H3K27Ac, and EZH2) quantified from immunoblots relative each corresponding loading control H4 or GAPDH, normalized to parental HT1080. Mean ± SEM is shown with individual replicates indicated by circles from two experiments with stable transfection (see Figs A, and A and F) and by squares from two experiments with transient transfection (24 and 48 h, see Fig B and G), n = 4. Student's t ‐test, ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. Original data for all quantifications, including P ‐values, are shown as source data. SWI/SNF opposes the polycomb complex PRC2. The catalytic subunit of PRC2, EZH2, catalyzes the trimethylation of Lys27 on histone H3 (H3K27me3), a chromatin modification associated with closed chromatin, leading to downregulation of polycomb‐regulated genes. FET fusion binding to SWI/SNF might compromise the function of SWI/SNF, including polycomb opposition. In particular, genes downregulated when FUS‐DDIT3 is overexpressed in HT1080 cells (RNA‐seq data, n = 3, compared to control, n = 4) overlap with upregulated gene‐sets after EZH2 knockdown, NIPP1 knockdown and HDAC knockdown, and BAF57 (SMARCE1) reconstitution in a SMARCE1 null cell line. RNA‐seq data of HT1080 wt ( n = 4), HT1080 EGFP ( n = 4), HT1080 FUS‐DDIT3‐EGFP ( n = 3), and HT1080 EWSR1‐FLI1‐EGFP ( n = 4) were generated and genes > twofold regulated were analyzed using the molecular signature database (MSigDB). Gene lists were compared to the gene‐set collection “chemical and genetic perturbations” and top 20 gene‐sets for each comparison are shown in the source data. q ‐values (FDR‐adjusted P ‐values) are indicated in parentheses. Schematic presentation of FET family of fusion oncoproteins, targeted DNA sequences, and associated tumors. Left: FET N‐terminal fusion partners and binding to SWI/SNF. Center: C‐terminal transcription factor fusion partners and their DNA target sequences (JASPAR database). Right: Tumor types caused by respective fusion oncogene. Note tumor type specificity for each fusion oncoprotein and that the FET‐NTDs replace each other as fusion partners in some entities. Only a selection of the FET family fusion oncoproteins and tumors is shown as more members are continuously discovered. Source data are available online for this figure.

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: Western Blot, Stable Transfection, Transfection, Expressing, Control, Modification, Binding Assay, RNA Sequencing, Knockdown, Generated, Comparison, Selection

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: A Immunoblot analysis (IB) of histone modifications in replicate R2 of stably transfected HT1080 cell lines expressing EGFP, FUS‐DDIT3‐EGFP, or EWSR1‐FLI1‐EGFP. Antibodies against H3K27Ac, H3K27me3, H3K4me3, and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount. Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion oncoproteins and EGFP. Stable replicate R1 is visualized in Fig A. B Immunoblot analysis (IB) of HT1080, and HT1080 transiently expressing FUS‐DDIT3‐EGFP or EWSR1‐FLI1‐EGFP (samples harvested after 24 or 48 h transient transfection) with antibodies against H3K27Ac, H3K27me3, H3K4me3, and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount. Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion proteins. C, D Immunoblot analysis (IB) of HT1080 and HT1080 FUS‐DDIT3‐EGFP (C) or HT1080 EWSR1‐FLI1‐EGFP (D) (both stable expression) with antibodies against H3K27me3, H3K4me3, and histone loading control H4. Immunoblot analysis with GFP or FLI1 antibody is shown to verify expression of FET fusion protein and normal FLI1. Note that endogenous FLI1 around 50 kDa is expressed in HT1080 cell lines. E Immunoblot analysis (IB) of extracts from untreated EWS TC‐71, cells treated with 5 μM tazemetostat for 72 h and DMSO‐control. Detection using antibodies against H3K27me3, H3K4me3, and histone loading control H4 showed substantial decrease of H3K27me3 after inhibition of EZH2 with tazemetostat. F Diagrams showing amount, quantified from immunoblots in Fig A (R1) or panel (A) (R2), of H3K27me3, H3K4me3, and H3K27Ac (in relation to corresponding histone loading control H4), ratio H3K27me3/H3K4me3 as well as amount of EZH2 compared to loading control GAPDH for HT1080 EGFP, HT1080 FUS‐DDIT3‐EGFP, and HT1080 EWSR1‐FLI1‐EGFP normalized to parental HT1080. Each diagram panel shows data from one experiment. G Diagrams showing amount, quantified from immunoblots in panel (B), of H3K27me3, H3K4me3, and H3K27Ac (in relation to corresponding histone loading control H4), ratio H3K27me3/H3K4me3 as well as amount of EZH2 compared to loading control GAPDH for HT1080 FUS‐DDIT3‐EGFP and HT1080 EWSR1‐FLI1‐EGFP normalized to parental HT1080. Samples were harvested after 24‐h or 48‐h transient transfection. Each diagram panel shows data from one experiment. H, I Diagram showing amount of H3K27me3 and H3K4me3 (in relation to corresponding histone loading control H4) as well as ratio H3K27me3/H3K4me3 for HT1080 FUS‐DDIT3‐EGFP (H) quantified from panel (C) or HT1080 EWSR1‐FLI1‐EGFP (I) quantified from panel (D) (both stable expression) normalized to parental HT1080. Each diagram panel shows data from one experiment. J Diagram showing amount, quantified from immunoblots in panel (E), of H3K27me3 and H3K4me3 (in relation to corresponding histone loading control H4) as well as ratio H3K27me3/H3K4me3 for untreated EWS TC‐71, cells treated with 5 μM tazemetostat for 72 h and DMSO‐control, normalized to the untreated control. Data from one experiment. Source data are available online for this figure.

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: Western Blot, Stable Transfection, Transfection, Expressing, Control, Inhibition

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: Immunoblot analysis (IB) of GFP‐tagged proteins co‐immunoprecipitated with BRG1. Detection with antibodies against BRG1 and DDIT3 in HT1080 cell lines with stable expression of FUS‐DDIT3‐EGFP or DDIT3‐EGFP. In order to directly quantify the fraction of bound and non‐bound protein, relative amounts of protein for each IP‐sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. One representative immunoblot is shown. Immunoblots from all replicates are shown in the source data. Graphs showing the percentage of bound to total (bound + non‐bound) signal intensities from immunoblots for DDIT3 in HT1080 FUS‐DDIT3‐EGFP or DDIT3‐EGFP. The specific interaction (BRG1) is compared to the non‐specific interaction (negative control IgG). Mean ± SEM is shown with individual replicates indicated by circles, n = 3. Student's t ‐test, ns = not significant. Original data for all quantifications, including P ‐values, are shown as source data. Source data are available online for this figure.

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: Western Blot, Immunoprecipitation, Expressing, Negative Control

Journal: EMBO Reports

Article Title: FET family fusion oncoproteins target the SWI / SNF chromatin remodeling complex

doi: 10.15252/embr.201845766

Figure Lengend Snippet: Verification of cell lines by RT–PCR over fusion breakpoints (brkp) of MLS 2645‐94 (FUS‐DDIT3 type II), MLS 402‐91 (FUS‐DDIT3 type I), EWS TC‐71 (EWSR1‐FLI1 type I), EWS IOR/CAR (EWSR1‐ERG), HT1080 FUS‐DDIT3‐EGFP (FUS‐DDIT3 type II), and HT1080 EWSR1‐FLI1‐EGFP (EWSR1‐FLI1 type I): list of cell line, fusion type, assay used (forward and reverse primer) and expected product size indicated.

Article Snippet: For the DDIT3 IP (DDIT3‐biotin antibody, NB600‐1335B, Novus Biologicals, Littleton, CO, USA) and the FLI1 IP (FLI1‐biotin antibody, US biologicals 246159‐biotin, Salem, MA, USA), the protocol was scaled up three times and samples were eluted in 2 × 75 μl or 2 × 150 μl 2x LDS sample buffer with 10% sample reducing agent.

Techniques: Reverse Transcription Polymerase Chain Reaction