mouse anti human brd4 antibody  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Phenotypic screening converges on CDK9 inhibition as a therapeutic strategy in translocation renal cell carcinoma

doi: 10.1101/2025.08.25.672235

Figure Lengend Snippet: (A) Outline of the chromatin displacement assay workflow. (B) Application of chromatin displacement assay to BRD4, using the tool compound JQ1. HEK293T cells were treated with JQ1 dose titration (3 nM to 10 μM). Cells were subject to in situ cell extraction (or not, bottom vs. top rows) and two color IF staining for BRD4 (green) and nucleus (red) was performed. Dose-dependent displacement of BRD4 from chromatin by JQ1 was quantified and plotted for each of 6 individual runs. Data are presented as mean ± SD. (C) Visualization of TFE3 staining and localization in HEK293T cells, with or without Torin1 treatment (which induces nuclear TFE3 localization), and with or without extraction. TFE3 cytoplasmic or nuclear intensity is quantified based on the IF images (mean ± SD, n=6). Note that cytoplasmic signal (white arrow) is dramatically reduced by extraction while nuclear (i.e. chromatin-bound) signal (blue arrow) is not. P -values computed by unpaired t-test; ****P<0.0001. (D) Visualization of ASPL-TFE3 fusion staining and localization in FUUR1 tRCC cells. Cytoplasmic and nuclear staining of the TFE3 fusion with or without extraction was quantified based on the IF images (mean ± SD, n=6) similar to panel (C). Note that TFE3 fusions are constitutively nuclear, as compared with WT TFE3, which shuttles between the cytoplasm and nucleus. P -values computed by unpaired t-test; *P<0.05, ****P<0.0001. (E) Composition of epigenetic modulator (“Epi-Mod”) compound library (n = 121 compounds) used for pilot screening in TFE3 chromatin displacement assay. (F) Replicate-Replicate scatterplot of Epi-Mod library screen using chromatin displacement assay in FUUR1 cells. Select inhibitor classes are highlighted: HDACi, green; HATi, red; HKMTi, blue. (G) Top, IF images showing displacement of ASPL-TFE3 fusion by panobinostat (10 μM) (with extraction condition). Staining: TFE3, green; Nucleus: Red. Quantification of dose-dependent displacement of TFE3, BRD4 and nuclear staining by panobinostat. Data is presented as mean ± SD, n=2.

Article Snippet: For immunofluorescence staining, the fixed cells were blocked (PBS + 1% BSA) for 30 minutes at room temperature and stained with primary antibodies for 1 hour at 37°C at the following dilutions: rabbit anti-human TFE3 antibody (Millipore Sigma #ZRB1272) at 1:1000 and mouse anti-human BRD4 antibody (Santa Cruz Biotechnology #sc518021) at 1:200.

Techniques: Titration, In Situ, Extraction, Staining, Drug discovery

Journal: bioRxiv

Article Title: Phenotypic screening converges on CDK9 inhibition as a therapeutic strategy in translocation renal cell carcinoma

doi: 10.1101/2025.08.25.672235

Figure Lengend Snippet: (A) Screening funnel of 25,000 compounds leading to 4 hits after dose response validation studies. (B) Replicate-Replicate scatterplot for run 4 (out of 5 total runs) of chromatin displacement screening of 25,000 compounds in in FUUR1 cells. Chromatin displacers, red; chromatin retention, green (see methods for hit selection from the 5 separate runs). Primary hits from this run are labeled in red or green; validated hits are named. (C) Dose-dependent increase in nuclear signal of TFE3 with BRD6866 treatment (chromatin retention) in a chromatin displacement assay (CDA) in FUUR1 cells. Nuclear signal (DAPI) as well as nuclear IF signal for TFE3 and BRD4 were quantified and plotted (mean ± SD, n=3). (D) Dose-dependent decrease in nuclear signal of TFE3 with BRD7659 (chromatin displacement) in a CDA in FUUR1 cells. Nuclear signal (DAPI) as well as nuclear IF signal for TFE3 and BRD4 were quantified and plotted (mean ± SD, n=3). (E) Chemical structure of BRD6866 and BRD7659. (F) RNA Seq of UOK109 cells after 16h treatment with BRD6866 at 1μM. Differential transcriptomics analysis using a volcano plot is shown. Antiapoptotic genes with known sensitivity to CDK9 inhibition, MCL1 and XIAP are labeled . RNA Seq schematic created using BioRender.com. (G) Hallmark gene set pathway enrichment analysis upon BRD6866 treatment was compared to two recent studies with CDK9 inhibition (CDK9i) and CDK9 degradation (CDK9d) ( , ). (H) CDK activity profiling assay (Reaction Biology) showing extent of inhibition of various CDK/cyclin pairs by BRD6866 (10 μM). Loss of activity for each CDK and cyclin pairs by BRD6866 was compared to DMSO control. CDK9/cyclin pairs are bolded. The cyclin interacting motif PFTAIRE defines a subgroup of CDKs that does not fall under named categories.

Article Snippet: For immunofluorescence staining, the fixed cells were blocked (PBS + 1% BSA) for 30 minutes at room temperature and stained with primary antibodies for 1 hour at 37°C at the following dilutions: rabbit anti-human TFE3 antibody (Millipore Sigma #ZRB1272) at 1:1000 and mouse anti-human BRD4 antibody (Santa Cruz Biotechnology #sc518021) at 1:200.

Techniques: Biomarker Discovery, Selection, Labeling, RNA Sequencing, Inhibition, Activity Assay, Control

Journal: Nature Communications

Article Title: Zfp260 choreographs the early stage osteo-lineage commitment of skeletal stem cells

doi: 10.1038/s41467-024-54640-0

Figure Lengend Snippet: a Workflow of in vivo labeling strategy using TAM and the experimental design. b PCA indicating the variations of transcriptomes among Lin - ZsGreen + cells isolated from BF-Ctrl, BF-cKO, MSFL-Ctrl and MSFL-cKO groups. c GO and KEGG enrichment analysis of FACS-RNA-seq data. d Heatmap of replicate data for H3K4me1 and H3K27ac enrichment as detected by CUT&Tag. n = 3 from 3 biological replicates. e GO-biological process enrichment analysis of differentially enriched super-enhancers. f Heatmap of replicate data for Zfp260-V5 enrichment detected by ChIP-seq. n = 2 from 2 biological replicates. g Top enriched de novo motifs of Zfp260-V5 enriched genes. h Distribution of peaks in the genome. i GO and KEGG enrichment analysis of Zfp260-V5 enriched genes. j Screening strategy for the potential master downstream regulator. k Transcripts Per Kilobase (TPM) of Runx2 expression level from fracture and MSFL derived Lin - ZsGreen + cells. n = 3 from 3 biological replicates of RNA-seq data. l Genome browser view of peaks enriched for H3K4me1, Brd4, H3K27ac, and Zfp260-V5 over the Runx2 gene locus on chromosome 17 (left) with the magnified super-enhancer region displayed on the right. Primers 1 and 2 indicated the primer sets for the subsequent ChIP-qPCR detection. m Co-IP was performed to examine the condensates for the super-enhancer via immortalized PSCs. n = 3 from 3 biological replicates. n , o mIHC co-staining for Zfp260 (purple) with Brd4 (gold), Med1 (cyan), and P300 (gray) in the homeostatic and osteogenic states of PSCs. The yellow dotted line indicated the route for the subsequent fluorescence intensity measurements. n = 3 from 3 biological replicates. p Fluorescence intensity measurements along the route, with black triangles indicating the merged signals of the four channels. q , r ChIP-qPCR assays for H3K27ac and Brd4 binding via immortalized PSCs. n = 6 from 2 biological replicates. Two-way ANOVA. Scalebars: 5 μm. All data in this figure are represented as mean ± SD. Source data and exact p values are provided in the Source Data file.

Article Snippet: The primary antibodies used in mIHC (dilution 1:400 for all antibodies) included goat anti-mouse/human/rat Itgav (AF1219, Novus Biologicals), mouse anti-mouse/rat CD90 (NB100-65543, Novus Biologicals), mouse anti-mouse/human CD105 (NBP2-22122, Novus Biologicals), rabbit anti-human/mouse/rat CD200 (AF2724, Novus Biologicals), rabbit anti-mouse/human/rat Runx2 (ab236639, Abcam), rabbit anti-mouse/human/rat Sox9 (ab185966, Abcam), rabbit anti-mouse/human Alpl (MA5-24845, Invitrogen), rabbit anti-mouse/human/rat Zfp260 (ABE295, Merck), mouse anti-human/mouse/rat p300 (NB100-616, Novus Biologicals), rabbit anti-human/mouse MED1 (NB100-2574, Novus Biologicals), rabbit anti-human/mouse BRD4 (NBP2-76393, Novus Biologicals), mouse anti-human/mouse/rat Prkca (NB600-201, Novus Biologicals), rabbit anti-V5 tag (13202, CST), mouse anti-Collagen type I (67288-1-Ig, proteintech), rabbit anti-Collagen type II (28459-1-AP, proteintech).

Techniques: In Vivo, Labeling, Isolation, RNA Sequencing, ChIP-sequencing, Expressing, Derivative Assay, ChIP-qPCR, Co-Immunoprecipitation Assay, Staining, Fluorescence, Binding Assay

Journal: Nature Communications

Article Title: Zfp260 choreographs the early stage osteo-lineage commitment of skeletal stem cells

doi: 10.1038/s41467-024-54640-0

Figure Lengend Snippet: a GST-pull down assay of PSC’s whole cell lysate (WCL). The red dotted box indicated the regions for M/S analysis. b GST-Zfp260 specially enriched kinases with high HT sequest scores. c Co-IP of 293 T cell line. d Co-IP of immortalized PSCs. e Co-staining image for Zfp260 and Prkca in PSCs. The white dotted circle indicated the nucleus (Left). The white dotted line indicated the route for the fluorescence intensity measurements (right). f Representative images of immunofluorescence of Zfp260 with osteogenic induction, with the MFI/cytosolic MFI calculated (right). g Separation of nuclear (NE) and cytosolic extracts (CE) followed by Western blot. h In vitro Phos-Assay was performed by Phospho-PAGE. i Co-IP, Phos-PAGE, and SDS-PAGE were jointly performed in immortalized PSCs. The red dotted rectangle indicating the Y173, S182, and S197 residues. j Suggested binding mode of Zfp260 and Prkca by AlphaFold2. k The shortest distance between Zfp260-aa173 and the catalytic domain (CD) of Prkca (Z173-CD), aa182 and CD (Z182-CD), and aa197 and CD (Z197-CD) were calculated. l Co-IP was performed in immortalized PSCs, with statistical analysis (right). m Representative images of immunofluorescence of Zfp260-V5 before and after osteogenic induction (left), with the nuclear MFI/cytosolic MFI evaluated (right). n Separation of NE and CE, followed by Western blot. o Representative ARS staining images of PSCs. Scalebar: 2 mm. p–r ChIP-qPCR assays for Zfp260-V5, Brd4, and H3K27ac binding. n = 6 from 2 biological replicates. For ( a , d , e , h , i ), experiments were conducted independently 3 times, consistently producing similar results. For ( c , g , h , i , l , n , o ), n = 3 from 3 biological replicates. For ( f , m ), n = 30 from 3 biological replicates, with 10 randomly selected cells calculated per replicate. For ( e , f , m ), scale bars: 5 μm. Two-way ANOVA. Box plots display the minimum and maximum values, with the center line representing the median, and the bounds of the box representing the 25th to 75th percentiles. Other data in this figure are represented as mean ± SD. Source data and exact p values are provided in the Source Data file.

Article Snippet: The primary antibodies used in mIHC (dilution 1:400 for all antibodies) included goat anti-mouse/human/rat Itgav (AF1219, Novus Biologicals), mouse anti-mouse/rat CD90 (NB100-65543, Novus Biologicals), mouse anti-mouse/human CD105 (NBP2-22122, Novus Biologicals), rabbit anti-human/mouse/rat CD200 (AF2724, Novus Biologicals), rabbit anti-mouse/human/rat Runx2 (ab236639, Abcam), rabbit anti-mouse/human/rat Sox9 (ab185966, Abcam), rabbit anti-mouse/human Alpl (MA5-24845, Invitrogen), rabbit anti-mouse/human/rat Zfp260 (ABE295, Merck), mouse anti-human/mouse/rat p300 (NB100-616, Novus Biologicals), rabbit anti-human/mouse MED1 (NB100-2574, Novus Biologicals), rabbit anti-human/mouse BRD4 (NBP2-76393, Novus Biologicals), mouse anti-human/mouse/rat Prkca (NB600-201, Novus Biologicals), rabbit anti-V5 tag (13202, CST), mouse anti-Collagen type I (67288-1-Ig, proteintech), rabbit anti-Collagen type II (28459-1-AP, proteintech).

Techniques: Pull Down Assay, Co-Immunoprecipitation Assay, Staining, Fluorescence, Immunofluorescence, Western Blot, In Vitro, SDS Page, Binding Assay, ChIP-qPCR