rabbit anti pol ii ntd antibody  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: RNA polymerase loss by nuclear rupture drives LMNA cardiomyopathy

doi: 10.64898/2026.04.03.716433

Figure Lengend Snippet: A) Immunofluorescence for bromouridine (BrU) after cardiomyocytes were cultured with BrU for 2 hr to label nascent RNAs. One binucleated cardiomyocyte per image, with nuclei zoomed in. Texts and icons below images indicate nuclear states. Scale bar: 5 μm. All data in , except , are from cardiomyocytes freshly isolated from mice at day 14 post tamoxifen. B) Nuclear BrU intensity by nuclear states after 1-hr BrU incubation. Density and box plots: BrU intensity distribution and interquartile range. Circles: mean intensity within biological replicates (color coded). Asterisks: P < 0.05 from t -tests on linear regression-estimated means with mouse-clustered standard errors. Underlying data: 244 intact nuclei from 4 WT mice, 275 intact, 111 ruptured, 104 resealed nuclei from 3 Lmna CKO mice. C) Relationship between nuclear BrU intensity and nuclear NLS–tdTomato intensity in all types of nuclei in Lmna CKO cardiomyocytes (519 nuclei from 3 mice). Line: simple linear regression fit with 95% confidence interval. R: Pearson correlation coefficient. P: t -test p -value on linear regression-estimated means with mouse-clustered standard errors. See for individual replicates. D) Immunofluorescence for RNA polymerase II (Pol II) in cardiomyocytes. Scale bar: 5 μm. E) Nuclear Pol II intensity by nuclear states. Underlying data: 285 intact nuclei from 3 WT mice, 180 intact, 133 ruptured, 136 resealed nuclei from 3 Lmna CKO mice. Graph annotations and statistics as in (B) . F) Relationship between Pol II intensity and NLS–tdTomato intensity in all types of nuclei in Lmna CKO cardiomyocytes (495 nuclei from 3 mice). Graph annotations and statistics as in (C) . G) Pol II ChIP-seq and input read coverage in WT and Lmna CKO cardiomyocytes (3 mice per genotype). ChIP-seq signals are normalized to internal spike-in controls. H) Average Pol II ChIP-seq signals across 21,177 protein-coding genes. X-axis: 100 equally-spaced bins in gene bodies, 5 bins for 1 kb-upstream regions, and 10 bins for 2 kb-downstream regions. I) Statistical comparison of gene-body Pol II signals in Lmna CKO versus WT cardiomyocytes for 11,942 Pol II-bound genes. Pol II-lost or gained genes are defined at limma p -value < 0.05. J) Ten most enriched Gene Ontology terms among the 1,759 Pol II-lost genes in Lmna CKO cardiomyocytes, with three representative genes for each term. P: Metascape p -value. K) Gene expression state of Pol II-lost, Pol II-gained, and all other genes in Lmna CKO (n=5) versus WT (n=7) hearts. P, DESeq2 p -value. RNA-seq data from En et al. 2024. L) Summary of . Nuclear rupture causes transcriptional deficiency due to RNA Pol II loss.

Article Snippet: Lysates were incubated with rabbit anti-Pol II NTD antibody (Cell Signaling Technology, #14958) overnight at 4 °C.

Techniques: Immunofluorescence, Cell Culture, Isolation, Incubation, ChIP-sequencing, Comparison, Gene Expression, RNA Sequencing

Journal: bioRxiv

Article Title: RNA polymerase loss by nuclear rupture drives LMNA cardiomyopathy

doi: 10.64898/2026.04.03.716433

Figure Lengend Snippet: A) Relationship between nuclear BrU intensity and nuclear NLS–tdTomato intensity in cardiomyocytes for each of 3 Lmna CKO mice. Line: simple linear regression fit with 95% confidence interval. R: Pearson’s correlation coefficient. All data in are derived from mice at day 14 post tamoxifen. B) Relationship between nuclear RNA Pol II intensity and nuclear NLS-tdTomato intensity in cardiomyocytes for each of 3 Lmna CKO mice. Other details as in (A) . C) Immunofluorescence for Pol II CTD phospho-Ser5 (Pol II Ser5p) in isolated cardiomyocytes expressing NLS-tdTomato and GFP-icGAS. Scale bar: 5 μm. D) Nuclear Pol II Ser5p intensity by nuclear states. Density and box plots (interquartile range): signal distribution of all nuclei. Circles: mean intensity within individual biological replicates (color coded). Asterisks: P < 0.05 from t-tests on linear regression-estimated means with mouse-clustered standard errors. Underlying data: 218 intact nuclei from 3 WT mice, 187 intact, 111 ruptured, 91 resealed nuclei from 3 Lmna CKO mice. E, F) Relationship between Pol II Ser5p intensity and NLS–tdTomato intensity in nuclei of Lmna CKO cardiomyocytes. (E) 424 nuclei from three biological replicates (mice). (F) Analysis within individual biological replicates. Other details as in (A) . G) Immunofluorescence for Pol II CTD phospho-Ser2 (Pol II Ser2p) in isolated cardiomyocytes expressing NLS-tdTomato and GFP-icGAS. Scale bar: 5 μm. H) Nuclear Pol II Ser2p intensity by nuclear states. Underlying data: 198 intact nuclei from 3 WT mice, 233 intact, 101 ruptured, 131 resealed nuclei from 3 Lmna CKO mice. N.S.: not significant. Other details as in (D) . I, J) Relationship between Pol II Ser2p intensity and NLS-tdTomato intensity in nuclei of Lmna CKO cardiomyocytes. (I) 517 nuclei from three biological replicates (mice). (J) Analysis within individual biological replicates. Other details as in (A) .

Article Snippet: Lysates were incubated with rabbit anti-Pol II NTD antibody (Cell Signaling Technology, #14958) overnight at 4 °C.

Techniques: Derivative Assay, Immunofluorescence, Isolation, Expressing

Journal: bioRxiv

Article Title: RNA polymerase loss by nuclear rupture drives LMNA cardiomyopathy

doi: 10.64898/2026.04.03.716433

Figure Lengend Snippet: A) Number of Pol II ChIP-seq and input sequencing reads aligned to the mouse genome (experimental) or the human genome (spike-in control). Scale factors are computed by spike-in control reads and sequencing depths and used to normalize Pol II ChIP-seq signals. All data in are derived from mice at 2 weeks post tamoxifen. B) Pol II ChIP-seq read coverage in all mouse genes stratified by gene-body Pol II coverage. Genes with Pol II coverage greater than or equal to 100 (2 in Log 10 ) were considered Pol II-bound (11,942 genes). C) Pol II ChIP-seq read coverage in all human genes, derived from the spike-in control chromatin. D) Gene-body Pol II ChIP-seq read coverage between every pair of biological replicates. E) Principal Component Analysis (PCA) of gene-body Pol II coverage in 11,942 Pol II-bound protein-coding genes. F) Cumulative fraction of 1,759 Pol II-lost genes and all other genes (y-axis) along the scale of differential gene expression between Lmna CKO hearts and wild-type hearts (x-axis). P, Kolmogorov-Smirnov test p -value comparing log 2 fold change of gene expression between Pol II-lost genes and all other genes. G) Gene expression state of Pol II-lost genes, Pol II-gained genes, and all other genes in the cardiomyocyte population in Lmna CKO (n=3) versus WT (n=3) hearts derived from single-nucleus RNA-seq in En et al. 2024. P, DESeq2 p -value. H) Same as F, but along the scale of differential gene expression between Lmna CKO and wild-type pseudo-bulk cardiomyocytes from the single-nucleus RNA-seq. P, Kolmogorov-Smirnov test p -value comparing log 2 fold change of gene expression between Pol II-lost genes and all other genes.

Article Snippet: Lysates were incubated with rabbit anti-Pol II NTD antibody (Cell Signaling Technology, #14958) overnight at 4 °C.

Techniques: ChIP-sequencing, Sequencing, Control, Derivative Assay, Gene Expression, RNA Sequencing

Journal: bioRxiv

Article Title: RNA polymerase loss by nuclear rupture drives LMNA cardiomyopathy

doi: 10.64898/2026.04.03.716433

Figure Lengend Snippet: A) Immunofluorescence for BANF1 and Desmin in human hearts. Top: heart biopsy from an individual with a LMNA p.R541C heterozygous mutation. Middle: autopsy heart from an individual with a pathogenic ACTC1 mutation. Bottom: apparently healthy autopsy heart. Inset: magnified areas enclosed by white squares. Scale bar: 20 μm. B) Three-dimensional reconstruction of BANF1 (green) and DNA (red) of nuclei (#1-4) in the LMNA p.R541C heart indicated in ( A ). Scale bar: 5 μm. C) Nuclei positive for nuclear-tip BANF1 per specimen field (normal heart, n=4 fields; ACTC1 heart, n=5 fields, LMNA -R541C heart, n=5 fields). Box: interquartile range. Asterisk: p -value <0.05 in Kruskal–Wallis test with Holm-corrected Wilcoxon tests. D) Model. Nuclear rupture drives LMNA -cardiomyopathy due to Pol II loss. Ruptured nuclei accumulate in mutant hearts due to frequent re-rupture of resealed nuclei.

Article Snippet: Lysates were incubated with rabbit anti-Pol II NTD antibody (Cell Signaling Technology, #14958) overnight at 4 °C.

Techniques: Immunofluorescence, Mutagenesis

Journal: Advanced Science

Article Title: A dsRNA Viral Transcriptional Regulator Evades Innate Immunity by Hijacking Host CoTranscription Factor DHX9

doi: 10.1002/advs.202512262

Figure Lengend Snippet: DHX9 is a host factor that interacts with σ3. (A) Purified proteins visualized by Coomassie blue staining. In HEK293T cells, Protein complexes associated with pNTAP‐σ3 were isolated by tandem affinity purification, separated by SDS‐PAGE, and visualized by Coomassie blue staining. Molecular weight marker is indicated on the left. The pNTAP empty vector served as a negative control. * indicates the bait protein σ3; (B) σ3 interacts with DHX9 in an DNA/RNA‐independent manner. HEK293T cells were transfected with either empty vector or pcDNA3.1‐Flag‐σ3. After 48 h, cells were harvested. Cell lysates were treated with or without RNase A (100 ng mL −1 ), Super Nuclease (25 U mL −1 ), or RNase III (10 U mL −1 ) at 37 °C for 20 min and then subjected for co‐IP; (C) The interaction between σ3 and DHX9 increases when cell lysates were treated with increasing amounts of poly I:C. Cell lysates from HEK293T cells were collected at 48 h post‐transfection and were then incubated with either poly I or poly I:C at the indicated concentrations on ice for 30 mins. After incubation, lysates were subjected for co‐IP using anti‐Flag agarose beads. (D) DHX9 interacts with σ3, but not σNS during REOV infection. HEK293T Cells were infected with REOV at a MOI of 10. At 18 hpi, cells were collected and lysed in the presence of Super Nuclease as described above. Co‐IP was then performed using anti‐DHX9 antibody. (E) σ3, but not µNS or σNS, specifically interacts with endogenous DHX9. (F) Subcellular localization of DHX9 or σ3 during the course of REOV infection. A549 cells were collected at 6 and 18 hpi, and then lysed for subcellular fractionation. Localization of indicated proteins in cytoplasm and nuclear fraction were assessed by western blotting. Histon H3 and Actin were used as marker for nuclear and cytoplasm fraction, respectively. (G) Relative band intensity was quantified for panel (F). Band intensities were all normalized to Mock sample. Data shown represent the mean ± s.d. of two independent experiments. Multiple non‐paired t test was used to analyze differences (ns = no significant, * p < 0.05). (H) DHX9 stays inside the nucleus in REOV infected cells. A549 cells were infected with REOV. At 18 h post infection, cells were fixed and co‐immunostained with rabbit anti‐DHX9 (in green) and mouse anti‐σ3 (in red). Yellow arrow indicates infected cells; white arrow indicates uninfected cells.

Article Snippet: Antibodies against RNA Pol II (Cell Signaling Technology, Cat No.14958, at 1:50 dilution), H3K4Me3 (Abcam, Cat No. ab12209, 1 μg), S9.6 (Absoluteantibody, Cat No. AB01137, with 1 μg), or IgG control (Cell Signaling Technology, Cat No.2729s, with 1 μg) were added to the cells, and incubated at 4 °C overnight, for S9.6 CUT & Tag, 10 μg μL −1 of RNase A was supplemented as negative control.

Techniques: Purification, Staining, Isolation, Affinity Purification, SDS Page, Molecular Weight, Marker, Plasmid Preparation, Negative Control, Transfection, Co-Immunoprecipitation Assay, Incubation, Infection, Fractionation, Western Blot

Journal: Advanced Science

Article Title: A dsRNA Viral Transcriptional Regulator Evades Innate Immunity by Hijacking Host CoTranscription Factor DHX9

doi: 10.1002/advs.202512262

Figure Lengend Snippet: Wild‐type σ3, but not K287T, impairs both the DHX9‐dependent recruitment and the pause‐release of RNA polymerase II (Pol II). (A) DHX9 helicase activity is required for the increased interaction between DHX9 and RNA Pol II upon TNF‐α treatment. Co‐IP experiment was performed in HEK293T between exogenously expressed Flag‐tagged DHX9 (either WT or K417R) and endogenous RNA pol II following TNF‐α treatment. The co‐IP efficiency was quantified by comparing the band intensity in the IP lane to the band in the input lane. All samples were normalized to mock (set at 1). Quantification of three independent experiments were shown in (B); (C) expression of σ3 WT, but not K287T, significantly reduced the interaction between DHX9 and RNA pol II upon TNF‐α treatment. The co‐IP efficiency was quantified by comparing the band intensity in the IP lane to the band in the input lane. All samples were normalized to mock (set at 1). Quantification of three independent experiments were shown in (D). (E) Average Pol II CUT & Tag signal profile on genomic loci (defined as 3 kb upstream of annotated TSS to 3 kb downstream of annotated TES) in HEK293T cells expressing σ3 or K287T upon TNF‐α treatment from two biological replicates. (F)Western blotting shows the equal expression levels of σ3(WT) and σ3(K287T). (G,H) Comparison of Counts per Million (CPM) of Pol II at promoter proximal region (TSS ± 500 bp) for (G) TNF‐α‐responsive genes ( n = 110) or (H) TNF‐α‐non‐responsive genes ( n = 896). Paired t ‐tests were used to evaluate the signal differences. (I,J) Pausing index of (I) TNF‐α‐responsive genes or (J) TNF‐α‐non‐responsive genes. (K‐M) ChIP‐qPCR analysis of Pol II enrichment at specific genomic regions (−1 kb from the TSS, ± 500 bp around TSS, and within the gene body) of RELB , CXCL3 , and ACTB in HEK293T cells expressing σ3 upon TNF‐α treatment. The condition labeled “σ3+RNase H” represents cells coexpressing σ3 and RNase H prior to TNF‐α treatment. Pol II enrichment was determined using the percent input method. Data are presented as the mean ± s.d. of three independent experiments. Paired t test was used to analyze the differences (ns = no significant, * p < 0.05, ** p < 0.01, *** p <0.001).

Article Snippet: Antibodies against RNA Pol II (Cell Signaling Technology, Cat No.14958, at 1:50 dilution), H3K4Me3 (Abcam, Cat No. ab12209, 1 μg), S9.6 (Absoluteantibody, Cat No. AB01137, with 1 μg), or IgG control (Cell Signaling Technology, Cat No.2729s, with 1 μg) were added to the cells, and incubated at 4 °C overnight, for S9.6 CUT & Tag, 10 μg μL −1 of RNase A was supplemented as negative control.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Expressing, Western Blot, Comparison, ChIP-qPCR, Labeling

Journal: Advanced Science

Article Title: A dsRNA Viral Transcriptional Regulator Evades Innate Immunity by Hijacking Host CoTranscription Factor DHX9

doi: 10.1002/advs.202512262

Figure Lengend Snippet: σ3 expression, leads to an upregulation of R‐loops at the promoter proximal region of TNF‐α‐responsive genes. (A) Average R‐loop (upper) and H3K4me3 (lower) CUT & Tag signal profile on genomic loci (defined as 3 kb upstream of annotated TSS to 3 kb downstream of annotated TES) in HEK293T cells expressing σ3 upon TNF‐α treatment. (B) Genome‐wide distribution of R‐loop (upper) and H3K4me3 (lower) peaks. (C and D) Comparison of abundance of R‐loop (C) and H3K4me3 (D) at promoter proximal region of TNF‐α‐responsive genes or TNF‐α‐non‐responsive genes. (E) Heatmaps show the read density that is ranked by decreasing occupancy of R‐loop (upper) and H3K4me3 (lower). (F,G) ChIP‐qPCR analysis of R‐loop (F) or H3K4me3 (G) enrichment at the promoter proximal region of RELB , CXCL3 , and ACTB in HEK293T cells expressing σ3 upon TNF‐α treatment. Enrichment is calculated by percent input method. Data are presented as the mean ± s.d. of three independent experiments. Paired t test was used to analyze the differences (ns = no significant, * p < 0.05, ** p < 0.01, *** p <0.001). (H) A working model of how σ3 inhibits NF‐κB gene expression (created using BioRender.com): ① σ3 competes with Pol II for interaction with DHX9, thereby decreasing the DHX9‐dependent recruitment of Pol II to NF‐κB‐dependent promoters. Meanwhile/subsequently, ② σ3 upregulates the R‐loop levels by inhibiting the helicase activity of DHX9, thereby affecting Pol II pause‐release and ultimately suppressing NF‐κB gene expression.

Article Snippet: Antibodies against RNA Pol II (Cell Signaling Technology, Cat No.14958, at 1:50 dilution), H3K4Me3 (Abcam, Cat No. ab12209, 1 μg), S9.6 (Absoluteantibody, Cat No. AB01137, with 1 μg), or IgG control (Cell Signaling Technology, Cat No.2729s, with 1 μg) were added to the cells, and incubated at 4 °C overnight, for S9.6 CUT & Tag, 10 μg μL −1 of RNase A was supplemented as negative control.

Techniques: Expressing, Genome Wide, Comparison, ChIP-qPCR, Gene Expression, Activity Assay

Journal: Nature Communications

Article Title: Monitoring rapid degradation of NANOG reveals UTP15 maintains pluripotency by regulating nascent transcripts

doi: 10.1038/s41467-025-67018-7

Figure Lengend Snippet: a Average reads density (top) and heatmap (bottom) analysis of ATAC-seq, H3K4me3, H3K27ac , UTP15 ChIP rep1, UTP15 ChIP rep2, and NANOG ChIP in the region from −2 kb upstream of TSS to +2 kb downstream of the TES across UTP15 target genes ( n = 685). Random genes were random selected from outside of UTP15 target genes ( n = 685). b Box plots showing normalized read counts (log₂) of UTP15 target genes ( n = 685 genes) from nascent RNA-seq in UTP15-AID (left panel) and NANOG-AID (right panel) cells treated with IAA. (two biological replicates). Box plots represent the median (centre line) and 25th-75th percentiles (bounds); whiskers extend to 1.5 × IQR. Two-sided Mann–Whitney U test. c IGV snapshot of UTP15 CLIP-seq, UTP15 ChIP-seq, NANOG ChIP-seq ( GSM4231200 ), Pol II ChIP-seq (UTP15-AID after treatment with IAA) and nascent RNA-seq at Zfp57, Zic5 or Zic2 gene locus. d Average reads density (top) and heatmap (bottom) analysis of UTP15 ChIP-seq at the TSSs across UTP15 target genes ( n = 685) after IAA induced NANOG degradation. Random genes were random selected from outside of UTP15 target genes ( n = 685). e Box plots represent UTP15 ChIP-seq signal intensity (log 2 ) at promoter regions of random genes (left, n = 685 genes) and UTP15 target genes (right, n = 685 genes) in NANOG-AID cells before (-IAA) and after (+IAA) NANOG degradation. Results are shown for two biological replicates (Rep1 and Rep2). Random genes, equal in number to the UTP15 target genes, were randomly selected from non-target regions. Box plots represent the median (centre line) and the 25th–75th percentiles (bounds); whiskers extend to 1.5 × IQR, and data points beyond the whiskers are considered outliers (not shown). Two-sided Mann-Whitney U test. f Western blot showing FLAG-mediated UTP15 co-IP in UTP15-AID cell lines under formaldehyde crosslinking conditions. The asterisk (*) denotes the purpose band. Representative western blots are shown from two independent experiments with consistent results. g Co-immunostaining of UTP15 and NANOG in UTP15-AID cells. Endogenous UTP15 protein was visualized using an anti-FLAG antibody. A magnified view of the indicated region is shown on the right. Scale bar, 5 μm. Representative immunofluorescence images are shown from two independent experiments with consistent results. Source data for Fig. 5f are provided as a Source Data file.

Article Snippet: Pol II S5P antibody (Abcam, AB5131), Pol II S2P antibody (Abcam, AB5095), Pol II NTD antibody (CST, 14958S).

Techniques: RNA Sequencing, MANN-WHITNEY, ChIP-sequencing, Western Blot, Co-Immunoprecipitation Assay, Immunostaining, Immunofluorescence

Journal: Nature Communications

Article Title: Monitoring rapid degradation of NANOG reveals UTP15 maintains pluripotency by regulating nascent transcripts

doi: 10.1038/s41467-025-67018-7

Figure Lengend Snippet: a Western blot showing HA-mediated UTP15 co-IP in POLR2A-FLAG knock-in cell lines. Representative western blots are shown from two independent experiments with consistent results. b Average read density analysis of Pol II ChIP-seq (NTD, Ser5, Ser2) after IAA induced acute UTP15 degradation in the region from −2 kb upstream of TSS to +2 kb downstream of the TES across UTP15 target genes ( n = 685). c Prediction of disorder regions for UTP15 using PONDR. The sequence of UTP15 was submitted to the PONDR server, which predicted regions of disorder with the VL-XT, VL3, VSL2 predictor. d Droplet formation assays of varying concentrations of GFP and GFP-UTP15 IDR in the presence of 16% dextran. Scale bar, 10 μm. e Quantification of nuclear UTP15 signal intensity in control and 1,6-HD treated cells (Ctrl: n = 18; 1,6-HD: n = 28, n represents individual nuclei analyzed). Cells were treated with 3% 1,6-HD for 10 minutes. Box plots show the median (centre line) and 25th-75th percentiles (bounds); whiskers extend to 1.5 × IQR. Statistical significance was assessed using a two-sided Mann-Whitney U test. f Representative immunofluorescence images of UTP15 (green) in UTP15-AID cells following treatment with 3% 1,6-HD for 10 min or control (untreated) conditions. Scale bar, 5 μm. g , h Droplet formation assays of mCherry-CTD (10 μM) with GFP (10 μM) or GFP-UTP15 IDR (10 μM) in the presence of 16% dextran. The incubation time was increased from 30 min to 150 min at room temperature. Quantification and representative pictures are shown in h and g , respectively. In h , y axis shows the sum of fluorescence intensity of mCherry-CTD within droplets in each field ( n = 5, n represents individual image analyzed, two-sided Student’s t -test). Scale bar, 10 μm. i Representative wide field and SIM² super-resolution images showing Pol II-NTD (green) and UTP15 (red) in UTP15-AID cells after IAA induced UTP15 degradation. Scale bar, 5 μm. j Quantification of nuclear Pol II clusters per cell using Laplace of Gaussian (LoG) filter method under different durations of IAA treatment ( n = 20 per group, n represents individual nuclei analyzed). Box plots show the median (centre line) and 25th-75th percentiles (bounds); whiskers extend to 1.5 × IQR. Statistical significance was assessed using a two-sided Mann-Whitney U test. Source data for Fig. 6a, e, h and j are provided as a Source Data file.

Article Snippet: Pol II S5P antibody (Abcam, AB5131), Pol II S2P antibody (Abcam, AB5095), Pol II NTD antibody (CST, 14958S).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Knock-In, ChIP-sequencing, Sequencing, Control, MANN-WHITNEY, Immunofluorescence, Incubation, Fluorescence

Journal: Nature Communications

Article Title: Monitoring rapid degradation of NANOG reveals UTP15 maintains pluripotency by regulating nascent transcripts

doi: 10.1038/s41467-025-67018-7

Figure Lengend Snippet: Acting as a master transcription factor, NANOG initiates the transcription of pluripotency genes. The newly synthesized RNA recruits UTP15 to chromatin at transcription start sites (TSSs). There, UTP15 supports the formation of transcription condensates by RNA polymerase II (Pol II). This UTP15-mediated process further amplifies and sustains the transcription of pluripotency genes.

Article Snippet: Pol II S5P antibody (Abcam, AB5131), Pol II S2P antibody (Abcam, AB5095), Pol II NTD antibody (CST, 14958S).

Techniques: Synthesized

Journal: Nature Communications

Article Title: Monitoring rapid degradation of NANOG reveals UTP15 maintains pluripotency by regulating nascent transcripts

doi: 10.1038/s41467-025-67018-7

Figure Lengend Snippet: a Average reads density (top) and heatmap (bottom) analysis of ATAC-seq, H3K4me3, H3K27ac , UTP15 ChIP rep1, UTP15 ChIP rep2, and NANOG ChIP in the region from −2 kb upstream of TSS to +2 kb downstream of the TES across UTP15 target genes ( n = 685). Random genes were random selected from outside of UTP15 target genes ( n = 685). b Box plots showing normalized read counts (log₂) of UTP15 target genes ( n = 685 genes) from nascent RNA-seq in UTP15-AID (left panel) and NANOG-AID (right panel) cells treated with IAA. (two biological replicates). Box plots represent the median (centre line) and 25th-75th percentiles (bounds); whiskers extend to 1.5 × IQR. Two-sided Mann–Whitney U test. c IGV snapshot of UTP15 CLIP-seq, UTP15 ChIP-seq, NANOG ChIP-seq ( GSM4231200 ), Pol II ChIP-seq (UTP15-AID after treatment with IAA) and nascent RNA-seq at Zfp57, Zic5 or Zic2 gene locus. d Average reads density (top) and heatmap (bottom) analysis of UTP15 ChIP-seq at the TSSs across UTP15 target genes ( n = 685) after IAA induced NANOG degradation. Random genes were random selected from outside of UTP15 target genes ( n = 685). e Box plots represent UTP15 ChIP-seq signal intensity (log 2 ) at promoter regions of random genes (left, n = 685 genes) and UTP15 target genes (right, n = 685 genes) in NANOG-AID cells before (-IAA) and after (+IAA) NANOG degradation. Results are shown for two biological replicates (Rep1 and Rep2). Random genes, equal in number to the UTP15 target genes, were randomly selected from non-target regions. Box plots represent the median (centre line) and the 25th–75th percentiles (bounds); whiskers extend to 1.5 × IQR, and data points beyond the whiskers are considered outliers (not shown). Two-sided Mann-Whitney U test. f Western blot showing FLAG-mediated UTP15 co-IP in UTP15-AID cell lines under formaldehyde crosslinking conditions. The asterisk (*) denotes the purpose band. Representative western blots are shown from two independent experiments with consistent results. g Co-immunostaining of UTP15 and NANOG in UTP15-AID cells. Endogenous UTP15 protein was visualized using an anti-FLAG antibody. A magnified view of the indicated region is shown on the right. Scale bar, 5 μm. Representative immunofluorescence images are shown from two independent experiments with consistent results. Source data for Fig. 5f are provided as a Source Data file.

Article Snippet: The following antibodies were used: FLAG (1:200, Sigma M2), Pol II NTD (1:100, CST 14958S), NANOG (1:100, Abcam), HA (1:100, Abcam).

Techniques: RNA Sequencing, MANN-WHITNEY, ChIP-sequencing, Western Blot, Co-Immunoprecipitation Assay, Immunostaining, Immunofluorescence

Journal: Nature Communications

Article Title: Monitoring rapid degradation of NANOG reveals UTP15 maintains pluripotency by regulating nascent transcripts

doi: 10.1038/s41467-025-67018-7

Figure Lengend Snippet: a Western blot showing HA-mediated UTP15 co-IP in POLR2A-FLAG knock-in cell lines. Representative western blots are shown from two independent experiments with consistent results. b Average read density analysis of Pol II ChIP-seq (NTD, Ser5, Ser2) after IAA induced acute UTP15 degradation in the region from −2 kb upstream of TSS to +2 kb downstream of the TES across UTP15 target genes ( n = 685). c Prediction of disorder regions for UTP15 using PONDR. The sequence of UTP15 was submitted to the PONDR server, which predicted regions of disorder with the VL-XT, VL3, VSL2 predictor. d Droplet formation assays of varying concentrations of GFP and GFP-UTP15 IDR in the presence of 16% dextran. Scale bar, 10 μm. e Quantification of nuclear UTP15 signal intensity in control and 1,6-HD treated cells (Ctrl: n = 18; 1,6-HD: n = 28, n represents individual nuclei analyzed). Cells were treated with 3% 1,6-HD for 10 minutes. Box plots show the median (centre line) and 25th-75th percentiles (bounds); whiskers extend to 1.5 × IQR. Statistical significance was assessed using a two-sided Mann-Whitney U test. f Representative immunofluorescence images of UTP15 (green) in UTP15-AID cells following treatment with 3% 1,6-HD for 10 min or control (untreated) conditions. Scale bar, 5 μm. g , h Droplet formation assays of mCherry-CTD (10 μM) with GFP (10 μM) or GFP-UTP15 IDR (10 μM) in the presence of 16% dextran. The incubation time was increased from 30 min to 150 min at room temperature. Quantification and representative pictures are shown in h and g , respectively. In h , y axis shows the sum of fluorescence intensity of mCherry-CTD within droplets in each field ( n = 5, n represents individual image analyzed, two-sided Student’s t -test). Scale bar, 10 μm. i Representative wide field and SIM² super-resolution images showing Pol II-NTD (green) and UTP15 (red) in UTP15-AID cells after IAA induced UTP15 degradation. Scale bar, 5 μm. j Quantification of nuclear Pol II clusters per cell using Laplace of Gaussian (LoG) filter method under different durations of IAA treatment ( n = 20 per group, n represents individual nuclei analyzed). Box plots show the median (centre line) and 25th-75th percentiles (bounds); whiskers extend to 1.5 × IQR. Statistical significance was assessed using a two-sided Mann-Whitney U test. Source data for Fig. 6a, e, h and j are provided as a Source Data file.

Article Snippet: The following antibodies were used: FLAG (1:200, Sigma M2), Pol II NTD (1:100, CST 14958S), NANOG (1:100, Abcam), HA (1:100, Abcam).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Knock-In, ChIP-sequencing, Sequencing, Control, MANN-WHITNEY, Immunofluorescence, Incubation, Fluorescence

Journal: Nature Communications

Article Title: Monitoring rapid degradation of NANOG reveals UTP15 maintains pluripotency by regulating nascent transcripts

doi: 10.1038/s41467-025-67018-7

Figure Lengend Snippet: Acting as a master transcription factor, NANOG initiates the transcription of pluripotency genes. The newly synthesized RNA recruits UTP15 to chromatin at transcription start sites (TSSs). There, UTP15 supports the formation of transcription condensates by RNA polymerase II (Pol II). This UTP15-mediated process further amplifies and sustains the transcription of pluripotency genes.

Article Snippet: The following antibodies were used: FLAG (1:200, Sigma M2), Pol II NTD (1:100, CST 14958S), NANOG (1:100, Abcam), HA (1:100, Abcam).

Techniques: Synthesized

Journal: Science Advances

Article Title: MeCP2 interacts with the super elongation complex to regulate transcription

doi: 10.1126/sciadv.adt5937

Figure Lengend Snippet: ( A ) Endogenous MeCP2 interacts with endogenous SEC subunits (AFF4, AF9, ENL, and ELL2) and RNA pol II in HEK293T cells. Normal mouse immunoglobulin G was used as a negative control. ( B ) Endogenous MeCP2 interacts with SEC subunits (AFF4 and ELL2) and RNA pol II in the cortex of WT mouse at 7 weeks of age. ( C ) Reverse IP of endogenous AFF4 from WT cortical lysate and immunoblotting against MeCP2. Different brightness settings were used for the top and bottom blots because of the relatively weaker MeCP2 co-IP band intensity compared to the AFF4 IP band intensity. Immunoblotting against AFF4 for (A) and (B) was performed with the Bethyl Laboratories antibody (A302-538A), whereas IP and immunoblotting for AFF4 for (C) was performed with the Proteintech antibody (14662-1-AP).

Article Snippet: Two micrograms of spike-in antibody (Active Motif, 61686) was added to each sample with one of the following antibodies for IPs: 5 μg of AFF4 antibody (Bosterbio, M03824), 5 μl of RNA pol II antibody (Cell Signaling Technology; 14958S), and 5 μg of pSer 2 RNA pol II antibody (EMD Millipore; 04-1571).

Techniques: Negative Control, Western Blot, Co-Immunoprecipitation Assay

Journal: Science Advances

Article Title: MeCP2 interacts with the super elongation complex to regulate transcription

doi: 10.1126/sciadv.adt5937

Figure Lengend Snippet: ( A ) Global heatmap of log 2 -transformed occupancy of AFF4 in the cortex of WT and Mecp2 null mice. ( B ) Global heatmap of log 2 fold change of AFF4 occupancy in Mecp2 null mouse compared to WT mouse. ( C ) Global heatmap of log 2 -transformed occupancy of RNA pol II in the cortex of WT and Mecp2 null mice. ( D ) Global heatmap of log 2 fold change of RNA pol II occupancy in Mecp2 null mouse compared to WT mouse. ( E ) Global heatmap of log 2 -transformed occupancy of pSer 2 RNA pol II in the cortex of WT and Mecp2 null mice. ( F ) Global heatmap of log 2 fold change of pSer 2 RNA pol II occupancy in Mecp2 null mouse compared to WT mouse. n = 8696 RNA pol II–bound genes are represented in all heatmaps.

Article Snippet: Two micrograms of spike-in antibody (Active Motif, 61686) was added to each sample with one of the following antibodies for IPs: 5 μg of AFF4 antibody (Bosterbio, M03824), 5 μl of RNA pol II antibody (Cell Signaling Technology; 14958S), and 5 μg of pSer 2 RNA pol II antibody (EMD Millipore; 04-1571).

Techniques: Transformation Assay

Journal: Science Advances

Article Title: MeCP2 interacts with the super elongation complex to regulate transcription

doi: 10.1126/sciadv.adt5937

Figure Lengend Snippet: ( A ) Global heatmap showing the log 2 fold change of AFF4, RNA pol II, and pSer 2 RNA pol II binding in the Mecp2 null cortex based on hierarchical clustering. ( B ) Two-dimensional plots for clusters in (A), showing the correlation between AFF4 and RNA pol II binding as the median of Mecp2 null versus WT ratios across matched animal pairs. n = 3 biological replicates. Color scale indicates the gene count. Spearman’s correlation values: ρ = 0.081; P = 0.00019 (cluster I), ρ = 0.13; P < 2.2 × 10 −16 (cluster II), ρ = 0.34; P = 4.8 × 10 −10 (cluster III). ( C ) Two-dimensional plots for clusters in (A), showing the correlation between AFF4 binding and RNA expression as the median of Mecp2 null versus WT ratios across matched animal pairs. Color scale indicates the gene count. n = 3 biological replicates for AFF4 ChIP-seq and n = 6 biological replicates for RNA-seq. Spearman’s correlation values: cluster I ρ = 0.026, P = 0.23; cluster II ρ = 0.063, P = 7.9 × 10 −07 ; cluster III ρ = 0.27, P = 5.3e-07.

Article Snippet: Two micrograms of spike-in antibody (Active Motif, 61686) was added to each sample with one of the following antibodies for IPs: 5 μg of AFF4 antibody (Bosterbio, M03824), 5 μl of RNA pol II antibody (Cell Signaling Technology; 14958S), and 5 μg of pSer 2 RNA pol II antibody (EMD Millipore; 04-1571).

Techniques: Binding Assay, RNA Expression, ChIP-sequencing, RNA Sequencing

Journal: iScience

Article Title: Targeting CDK7/12/13 functional synergism reverses myofibroblast activation and ameliorates lung fibrosis

doi: 10.1016/j.isci.2025.112778

Figure Lengend Snippet: The functional synergism of CDK7/12/13 is the critical determinant of lung myofibroblast activation (A) Heatmap showing relative expression of pro-fibrotic genes in TGFβ-induced lung myofibroblasts transfected with siRNA as indicated. Data are presented as a fold change when normalized to pro-fibrotic gene transcript levels in serum free media (SFM) starved HLFs transfected with control siRNA. (B) COL1A1 mRNA level in SFM starved HLFs and TGFβ-induced lung myofibroblasts transfected with siRNAs as indicated. (C) Representative images of αSMA immunofluorescence in SFM starved HLFs and TGFβ-induced lung myofibroblasts transfected with siRNAs as indicated. Scale bar: 50 μm. (D) Quantification of immunofluorescent intensity of αSMA in (C). (E) Representative images of a 24-well plate showing gel contraction in collagen matrices seeded with SFM starved HLFs and TGFβ-induced lung myofibroblasts transfected with siRNAs as indicated. The scale bar is the same as in (C) although not specifically indicated. (F) Quantification of collagen gel contraction in (E). Data are presented as percentage (%) of gel contraction-induced by TGFβ induced lung myofibroblasts transfected with control siRNA, which was arbitrarily set as 100%. (G) Representative western blot of nuclear RNA Pol II CTD S2P, S5P, S7P and total nuclear RNA Pol II in SFM starved HLFs and TGFβ-induced lung myofibroblasts transfected with siRNAs as indicated. Yin Yang 1 (YY1) is a loading control for nuclear extract. Data in (B), (D) and (F) represent mean ± S.E. of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001. p value is calculated using the unpaired two-tailed t-test.

Article Snippet: Equal amounts of protein lysates were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and subjected to immunoblotting analysis using following primary antibodies: anti-YY1 (46395, Cell Signaling Technology), anti-RNA Pol II (14958, Cell Signaling Technology), anti-S2P (13499, Cell Signaling Technology), anti-S5P (13523, Cell Signaling Technology), and anti-S7P (13780, Cell Signaling Technology).

Techniques: Functional Assay, Activation Assay, Expressing, Transfection, Control, Immunofluorescence, Western Blot, Two Tailed Test

Journal: iScience

Article Title: Targeting CDK7/12/13 functional synergism reverses myofibroblast activation and ameliorates lung fibrosis

doi: 10.1016/j.isci.2025.112778

Figure Lengend Snippet: Assessment of the effects of CDK7/12/13 inhibition on lung myofibroblast activation and proliferation in vivo (A) Representative immunofluorescent images of αSMA, RNA Pol II CTD S2P and S5P in lung tissues. Scale bar: 20 μm. (B–F) Quantification of percentage of αSMA + (B), S2P+ (C), S5P+ (D), S2P+/αSMA + (E) and S5P+/αSMA + (F) cells per field. (G) Representative immunofluorescent images of KI67 and αSMA in lung tissues. Scale bar: 20 μm. (H and I) Quantification of percentage of KI67+ cells (H) and KI67+/αSMA + cells(I). Data in (B–F) and (H–I) represent the mean value of five random fields per image. Data represent mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. p value is calculated using one-way ANOVA.

Article Snippet: Equal amounts of protein lysates were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and subjected to immunoblotting analysis using following primary antibodies: anti-YY1 (46395, Cell Signaling Technology), anti-RNA Pol II (14958, Cell Signaling Technology), anti-S2P (13499, Cell Signaling Technology), anti-S5P (13523, Cell Signaling Technology), and anti-S7P (13780, Cell Signaling Technology).

Techniques: Inhibition, Activation Assay, In Vivo

Journal: iScience

Article Title: Targeting CDK7/12/13 functional synergism reverses myofibroblast activation and ameliorates lung fibrosis

doi: 10.1016/j.isci.2025.112778

Figure Lengend Snippet: The functional synergism of CDK7/12/13 is the critical determinant of lung myofibroblast activation (A) Heatmap showing relative expression of pro-fibrotic genes in TGFβ-induced lung myofibroblasts transfected with siRNA as indicated. Data are presented as a fold change when normalized to pro-fibrotic gene transcript levels in serum free media (SFM) starved HLFs transfected with control siRNA. (B) COL1A1 mRNA level in SFM starved HLFs and TGFβ-induced lung myofibroblasts transfected with siRNAs as indicated. (C) Representative images of αSMA immunofluorescence in SFM starved HLFs and TGFβ-induced lung myofibroblasts transfected with siRNAs as indicated. Scale bar: 50 μm. (D) Quantification of immunofluorescent intensity of αSMA in (C). (E) Representative images of a 24-well plate showing gel contraction in collagen matrices seeded with SFM starved HLFs and TGFβ-induced lung myofibroblasts transfected with siRNAs as indicated. The scale bar is the same as in (C) although not specifically indicated. (F) Quantification of collagen gel contraction in (E). Data are presented as percentage (%) of gel contraction-induced by TGFβ induced lung myofibroblasts transfected with control siRNA, which was arbitrarily set as 100%. (G) Representative western blot of nuclear RNA Pol II CTD S2P, S5P, S7P and total nuclear RNA Pol II in SFM starved HLFs and TGFβ-induced lung myofibroblasts transfected with siRNAs as indicated. Yin Yang 1 (YY1) is a loading control for nuclear extract. Data in (B), (D) and (F) represent mean ± S.E. of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001. p value is calculated using the unpaired two-tailed t-test.

Article Snippet: Rabbit monoclonal anti-RNA Pol II , Cell Signaling Technology , Cat#14958; RRID: AB_2687876.

Techniques: Functional Assay, Activation Assay, Expressing, Transfection, Control, Immunofluorescence, Western Blot, Two Tailed Test

Journal: iScience

Article Title: Targeting CDK7/12/13 functional synergism reverses myofibroblast activation and ameliorates lung fibrosis

doi: 10.1016/j.isci.2025.112778

Figure Lengend Snippet: Assessment of the effects of CDK7/12/13 inhibition on lung myofibroblast activation and proliferation in vivo (A) Representative immunofluorescent images of αSMA, RNA Pol II CTD S2P and S5P in lung tissues. Scale bar: 20 μm. (B–F) Quantification of percentage of αSMA + (B), S2P+ (C), S5P+ (D), S2P+/αSMA + (E) and S5P+/αSMA + (F) cells per field. (G) Representative immunofluorescent images of KI67 and αSMA in lung tissues. Scale bar: 20 μm. (H and I) Quantification of percentage of KI67+ cells (H) and KI67+/αSMA + cells(I). Data in (B–F) and (H–I) represent the mean value of five random fields per image. Data represent mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. p value is calculated using one-way ANOVA.

Article Snippet: Rabbit monoclonal anti-RNA Pol II , Cell Signaling Technology , Cat#14958; RRID: AB_2687876.

Techniques: Inhibition, Activation Assay, In Vivo