Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) 抗体

• ChIP

Unknown

ChIP - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (AB5131)

Chromatin was prepared from U-2 OS (Human bone osteosarcoma epithelial cell line) cells according to the Abcam X-ChIP protocol.

Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 μg of chromatin, 2 μg of ab5131 (blue), and 20 μl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the inactive AFM and F8 promoters, the GAPDH promoter (active) and over the y-Actin gene (active).

Schematic diagram of the y-Actin gene is shown on the top of the figure. Black boxes represent exons and thin lines represent introns. PCR products are depicted as bars under the gene.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (AB5131)

ab5131 staining RNA polymerase II CTD repeat YSPTSPS (phospho S5) in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab5131 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (AB5131)

ICC/IF image of ab5131 stained HeLa(Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed in methanol for 5 minutes, permabilized in TBS-T for 20 minutes and incubated with the antibody (ab5131, 1 μg/ml) for 1 hour at room temperature. 1% BSA / 10% normal goat serum / 0.3M glycine was used to quench auto-fluorescence and block non-specific protein-protein interactions.

The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1 hour. Alexa Fluor^® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

• ChIP

PubMed

ChIP - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (AB5131)

Chromatin from cultured cells, mouse PVN punches (individual pools formed from groups of 3) or whole hypothalami dissected from fresh brains were cross linked, disrupted by sonification and purified.

The binding of Suz12 to wingless (Wnt1), a beta-catenin dependent developmental regulator and to the RNA polymerase II promoter (RNAPII), a housekeeping gene, served as positive and negative controls, respectively, in these experiments.

Murgatroyd et al PLoS One. 2014 Mar 5;9(3):e90277. doi: 10.1371/journal.pone.0090277. eCollection 2014. Fig S2. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• WB

Project

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (AB5131)

All lanes:

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (ab5131) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 20 µg

Lane 2:

S.cerevisiae (Y190) Whole Cell Lysate at 20 µg

Lane 3:

HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 20 µg with <em>S. cerevisiae</em> RNA polymerase II CTD repeat YSPTSPS peptide (<a href='/products/unavailable/s-cerevisiae-rna-polymerase-ii-ctd-repeat-ysptsps-peptide-ab12795'>ab12795</a>)

Lane 4:

S.cerevisiae (Y190) Whole Cell Lysate at 20 µg with <em>S. cerevisiae</em> RNA polymerase II CTD repeat YSPTSPS peptide (<a href='/products/unavailable/s-cerevisiae-rna-polymerase-ii-ctd-repeat-ysptsps-peptide-ab12795'>ab12795</a>)

Lane 5:

HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 20 µg with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (<a href='/products/proteins-peptides/human-rna-polymerase-ii-ctd-repeat-ysptsps-phospho-s5-peptide-ab18488'>ab18488</a>)

Lane 6:

S.cerevisiae (Y190) Whole Cell Lysate at 20 µg with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (<a href='/products/proteins-peptides/human-rna-polymerase-ii-ctd-repeat-ysptsps-phospho-s5-peptide-ab18488'>ab18488</a>)

Lane 7:

HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 20 µg with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide (<a href='/products/proteins-peptides/s-cerevisiae-rna-polymerase-ii-ctd-repeat-ysptsps-phospho-s2-peptide-ab12793'>ab12793</a>)

Lane 8:

S.cerevisiae (Y190) Whole Cell Lysate at 20 µg with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide (<a href='/products/proteins-peptides/s-cerevisiae-rna-polymerase-ii-ctd-repeat-ysptsps-phospho-s2-peptide-ab12793'>ab12793</a>)

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 217 kDa

true

Exposure time: 30s

• WB

AbReview40285****

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (AB5131)

All lanes:

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (ab5131) at 1/5000 dilution

Lane 1:

Xenopus laevis whole tissue lysate treated with DMSO for 24 hours

Lane 2:

Xenopus laevis whole tissue lysate treated with CDK inhibitor for 24 hours

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG polyclonal at 1/10000 dilution

Predicted band size: 217 kDa

Observed band size: 240 kDa

true

Exposure time: 1min

This image is courtesy of an anonymous Abreview

• WB

Unknown

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (AB5131)

Lanes 1, 3 and 4:

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (ab5131) at 1/500 dilution

Lane 2:

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (ab5131) at 1/2000 dilution

Lanes 1 - 2:

HeLa nuclear extract at 20 µg

Lane 3:

HeLa nuclear extract at 20 µg with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (<a href='/products/proteins-peptides/human-rna-polymerase-ii-ctd-repeat-ysptsps-phospho-s5-peptide-ab18488'>ab18488</a>)

Lane 4:

HeLa nuclear extract at 20 µg with <em>S. cerevisiae</em> RNA polymerase II CTD repeat YSPTSPS peptide (<a href='/products/unavailable/s-cerevisiae-rna-polymerase-ii-ctd-repeat-ysptsps-peptide-ab12795'>ab12795</a>)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab6721'>ab6721</a>) at 1/5000 dilution

Predicted band size: 217 kDa

Observed band size: 250 kDa

false

Exposure time: 30s

• WB

CiteAb

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (AB5131)

Western Blotting using Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody, ab5131. Publication image from Boutros, M. et al., 2020, Nat Commun, 32382029. Legend direct from paper.

dBRD4 promotes transcription elongation of NSL target genes.a Western blot for dBRD4 and H3 after 100 nM dBET6 (lanes 2 and 5) or 5 µM JQ1 (lanes 3  and 6) treatment. Blue asterisks indicate dBRD4-S and dBRD4-L. The experiment was repeated twice showing similar results. b–d Firefly luciferase activity using NSL3 and KANSL3 (human orthologue of NSL3) fused to Gal4 DNA-binding domain or full-length Gal4 to drive expression of the UAS-firefly luciferase reporter upon (b) 5 µM JQ1, (c) 1 µM iBET 762 or (d) 100 nM dBET6 treatment. Bars represent mean values ± SEM (n = 3 technical replicates). e Heatmap of total RNA-seq. Log2 fold changes of gene expression in NSL1 and NSL3 RNAi and JQ1 (5 µM) treatments for 1 h or 4 h versus control RNAi (GST) or DMSO for all expressed genes are plotted. Gene order was generated by unsupervised hierarchical clustering. f Boxplot of normalized RNA-seq counts in NSL1, dBRD4, dBRD4-L and control RNAi (GST) for NSL complex-bound genes (n = 5600). Two-sided Welch two sample t-test was applied. Boxplots show median (centre), interquartile-range (box) and minima/maxima (whiskers). g Scatterplot of gene expression changes after 4 h JQ1 treatment and NSL1 RNAi. Log2 fold changes for all genes are plotted. Linear regression model was applied. h Representative western blot for Pol2 ser2p, Pol2 ser5p and Rpb3 after NSL1 RNAi (lanes 3 and 4), for quantification see Supplementary Fig. 3j. H4 blot shown here is identical to a. i Representative western blot for Pol2 ser2p, Pol2 ser5p, Rpb3 and H3 after 100 nM dBET6 (lanes 2 and 5) or 5 µM JQ1 (lanes 3 and 6) treatment, for quantification see Supplementary Figure 3j. j. Average profiles of Pol2 (Rpb3) ChIP-seq signal for expressed NSL-bound (n = 5600) and expressed non-NSL-bound (n = 1600) genes after 1 h JQ1 (5 µM), 1 h dBET6 (100 nM) or NSL1 RNAi13 compared to controls (DMSO and GFP RNAi). Gene bodies are scaled from 0.5 kb until TESs. Drosophila virilis chromatin was added to experimental Drosophila melanogaster chromatin before Rpb3 ChIPs to control for IP efficiency and library composition effects (see Methods). e–g Expression was normalized using synthetic ERCC spikes (see Methods). n = 3 biological replicates. Source data for b–d are provided as a Source Data file.

false

• WB

CiteAb

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (AB5131)

Western Blotting using Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody, ab5131. Publication image from Boutros, M. et al., 2020, Nat Commun, 32382029. Legend direct from paper.

dBRD4 promotes transcription elongation of NSL target genes.a Western blot for dBRD4 and H3 after 100 nM dBET6 (lanes 2 and 5) or 5 µM JQ1 (lanes 3  and 6) treatment. Blue asterisks indicate dBRD4-S and dBRD4-L. The experiment was repeated twice showing similar results. b–d Firefly luciferase activity using NSL3 and KANSL3 (human orthologue of NSL3) fused to Gal4 DNA-binding domain or full-length Gal4 to drive expression of the UAS-firefly luciferase reporter upon (b) 5 µM JQ1, (c) 1 µM iBET 762 or (d) 100 nM dBET6 treatment. Bars represent mean values ± SEM (n = 3 technical replicates). e Heatmap of total RNA-seq. Log2 fold changes of gene expression in NSL1 and NSL3 RNAi and JQ1 (5 µM) treatments for 1 h or 4 h versus control RNAi (GST) or DMSO for all expressed genes are plotted. Gene order was generated by unsupervised hierarchical clustering. f Boxplot of normalized RNA-seq counts in NSL1, dBRD4, dBRD4-L and control RNAi (GST) for NSL complex-bound genes (n = 5600). Two-sided Welch two sample t-test was applied. Boxplots show median (centre), interquartile-range (box) and minima/maxima (whiskers). g Scatterplot of gene expression changes after 4 h JQ1 treatment and NSL1 RNAi. Log2 fold changes for all genes are plotted. Linear regression model was applied. h Representative western blot for Pol2 ser2p, Pol2 ser5p and Rpb3 after NSL1 RNAi (lanes 3 and 4), for quantification see Supplementary Fig. 3j. H4 blot shown here is identical to a. i Representative western blot for Pol2 ser2p, Pol2 ser5p, Rpb3 and H3 after 100 nM dBET6 (lanes 2 and 5) or 5 µM JQ1 (lanes 3 and 6) treatment, for quantification see Supplementary Figure 3j. j. Average profiles of Pol2 (Rpb3) ChIP-seq signal for expressed NSL-bound (n = 5600) and expressed non-NSL-bound (n = 1600) genes after 1 h JQ1 (5 µM), 1 h dBET6 (100 nM) or NSL1 RNAi13 compared to controls (DMSO and GFP RNAi). Gene bodies are scaled from 0.5 kb until TESs. Drosophila virilis chromatin was added to experimental Drosophila melanogaster chromatin before Rpb3 ChIPs to control for IP efficiency and library composition effects (see Methods). e–g Expression was normalized using synthetic ERCC spikes (see Methods). n = 3 biological replicates. Source data for b–d are provided as a Source Data file.

false

• WB

CiteAb

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (AB5131)

Western Blotting using Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody, ab5131. Publication image from Boutros, M. et al., 2020, Nat Commun, 32382029. Legend direct from paper.

dBRD4 promotes transcription elongation of NSL target genes.a Western blot for dBRD4 and H3 after 100 nM dBET6 (lanes 2 and 5) or 5 µM JQ1 (lanes 3  and 6) treatment. Blue asterisks indicate dBRD4-S and dBRD4-L. The experiment was repeated twice showing similar results. b–d Firefly luciferase activity using NSL3 and KANSL3 (human orthologue of NSL3) fused to Gal4 DNA-binding domain or full-length Gal4 to drive expression of the UAS-firefly luciferase reporter upon (b) 5 µM JQ1, (c) 1 µM iBET 762 or (d) 100 nM dBET6 treatment. Bars represent mean values ± SEM (n = 3 technical replicates). e Heatmap of total RNA-seq. Log2 fold changes of gene expression in NSL1 and NSL3 RNAi and JQ1 (5 µM) treatments for 1 h or 4 h versus control RNAi (GST) or DMSO for all expressed genes are plotted. Gene order was generated by unsupervised hierarchical clustering. f Boxplot of normalized RNA-seq counts in NSL1, dBRD4, dBRD4-L and control RNAi (GST) for NSL complex-bound genes (n = 5600). Two-sided Welch two sample t-test was applied. Boxplots show median (centre), interquartile-range (box) and minima/maxima (whiskers). g Scatterplot of gene expression changes after 4 h JQ1 treatment and NSL1 RNAi. Log2 fold changes for all genes are plotted. Linear regression model was applied. h Representative western blot for Pol2 ser2p, Pol2 ser5p and Rpb3 after NSL1 RNAi (lanes 3 and 4), for quantification see Supplementary Fig. 3j. H4 blot shown here is identical to a. i Representative western blot for Pol2 ser2p, Pol2 ser5p, Rpb3 and H3 after 100 nM dBET6 (lanes 2 and 5) or 5 µM JQ1 (lanes 3 and 6) treatment, for quantification see Supplementary Figure 3j. j. Average profiles of Pol2 (Rpb3) ChIP-seq signal for expressed NSL-bound (n = 5600) and expressed non-NSL-bound (n = 1600) genes after 1 h JQ1 (5 µM), 1 h dBET6 (100 nM) or NSL1 RNAi13 compared to controls (DMSO and GFP RNAi). Gene bodies are scaled from 0.5 kb until TESs. Drosophila virilis chromatin was added to experimental Drosophila melanogaster chromatin before Rpb3 ChIPs to control for IP efficiency and library composition effects (see Methods). e–g Expression was normalized using synthetic ERCC spikes (see Methods). n = 3 biological replicates. Source data for b–d are provided as a Source Data file.

false