ctd ser2p  (DIAGENODE DIAGNOSTICS)

Journal: Cell reports

Article Title: Multiple direct and indirect roles of the Paf1 complex in transcription elongation, splicing, and histone modifications

doi: 10.1016/j.celrep.2024.114730

Figure Lengend Snippet: (A and B) Browser tracks showing 4tU-seq read density over RPS14B . Dotted line denotes intron. (C) Diagram depicting the calculation of fraction of unspliced reads over the 5′ splice junction. (D and E) Violin plots of log 2 -fold changes in the fraction of unspliced reads at 5′ splice junctions for Paf1CΔ (D) or Paf1C-AID strains treated with auxin (30 min) (E). (F) Heatmap showing log 2 -fold change in fraction of unspliced reads over 5′ splice sites in ribosomal protein (RP) and non-RP genes sorted by fold effect of paf1Δ condition. (G and H) Metaplots of RNAPII-Ser2P ChIP-seq signal over protein coding genes. Significance determined by Mann-Whitney U test of control and experimental data: * p < 0.05, ** p < 0.01, and *** p < 0.001. See also .

Article Snippet: Mouse α-RNAPII CTD Ser2P (clone 3E10) , Active Motif , Cat# 91115; RRID: AB_2793780.

Techniques: ChIP-sequencing, MANN-WHITNEY, Control

Journal: Cell reports

Article Title: Multiple direct and indirect roles of the Paf1 complex in transcription elongation, splicing, and histone modifications

doi: 10.1016/j.celrep.2024.114730

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse α-RNAPII CTD Ser2P (clone 3E10) , Active Motif , Cat# 91115; RRID: AB_2793780.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Western Blot, Spot Test, Software

Journal: Nucleic Acids Research

Article Title: BOD1L mediates chromatin binding and non-canonical function of H3K4 methyltransferase SETD1A

doi: 10.1093/nar/gkae605

Figure Lengend Snippet: BOD1L loss decreases SETD1A on chromatin. ( A ) Browser view of ChIP-seq results against SETD1A, NELFE, RNAP2 NTD, Ser5P, Ser2P, H3K4me3 and H3K36me3 at INTS2 loci in sgBOD1L-expressing leukemia cells. ( B ) Average ChIP-seq signals of SETD1A, NELFE, NTD and Ser5P along DR (SETD1A-target) genes and flanking regions from sgAAVS and sgBOD1L-expressing cells are shown. ( C ) Violin plot indicating signal intensity of SETD1A at DR genes. ( D ) Pausing index (Log 2 ) of all genes and BOD1L/SETD1A targets. ( E ) Browser view of ChIP-seq results against SETD1A and BOD1L (top); 8307 peaks were overlapped (bottom). ( F ) Distributions of SETD1A, NTD and Ser5P at 9290 peaks ± 2kb harboring BOD1L binding 1 h after BOD1L degradation. Violin plot indicating signal intensities of each peak. ( G ) Distributions and signal intensities of H3K4me3 at BOD1L-binding regions 24 h after BOD1L degradation. In (C), (D), (F) and (G), data are presented as mean ± SD. ** P < 0.01. * P < 0.05. ns, no significance.

Article Snippet: Primary antibodies against HA-tag (#3724, Cell Signaling), SETD1A (#61702, Cell Signaling), RNAP2-NTD (#14958, Cell Signaling), RNAP2-Ser5P (#13523, Cell Signaling), RNAP2-Ser2P (#13499, Cell Signaling), NELFE (#ab170104, Abcam), H3K4me3 (#ab8580, Abcam), H3K36me3 (#9050, Abcam), RPA2 (#ab10359, Abcam), and RBBP5 (A300-109A, Bethyl Laboratories) were used.

Techniques: ChIP-sequencing, Expressing, Binding Assay

Journal: bioRxiv

Article Title: Fine tuning of Rpb4 phosphorylation modulates Rpb4/7 stoichiometry within yeast RNA polymerase II and regulates gene expression

doi: 10.1101/2023.12.15.571801

Figure Lengend Snippet: (A) The Rpb4-P level was specifically increased in the phosphatase mutant, fcp1-5001 . Left, WCE from wt and the CTD phosphatase mutants rtr1Δ , ssu72-2 , and fcp1-5001 were run in SDS-PAGE gels containing Phos-tag and submitted to western blotting with anti-Rpb4 to examine levels of Rpb4-P and non-phosphorylated Rpb4. Right, plots of the Rpb4-P/Rpb4 ratio determined by quantitation (by densitometry) of immunoreactive bands from the western blots. The mean values and standard errors are from three independent experiments. ns, not significant; ** p ≤ 0.01. (B) The Rpb4-P level is due to the phosphorylation of all, some, or any of the five phospho-residues mutated in rpb4-S/T-A . Analysis of the Rpb4-P level in wt , fcp1-5001 , rpb4-S/T-A , and fcp1-5001 rpb4-S/T-A double mutant cells as in (A). (C) Co-IP assays to analyze RNAPII integrity in wt and fcp1-5001 cells. WCE from wt and fcp1-5001 were used to immunoprecipitate either Rpb3 or Rpb4 using the corresponding antibodies. Immunoprecipitated samples were tested by western blot to detect Rpb1, Rpb1-Ser2P, Rpb4-TAP, and Rpb3. (D) ChIP-qPCR assays to evaluate Rpb3 and Rpb4 association with the promoter (P), coding (CD), and 3′-end (3′) regions of several constitutively transcribed genes. The black bars represent wt levels set up as 1 for both Rpb3 and Rpb4 association. Anti-Rpb3 or anti-Rpb4 were used to immunoprecipitate both RNAPII subunits and immunoprecipitated DNA was tested by qPCR. *** p ≤ 0.001.

Article Snippet: Western blot analysis was performed using the appropriate antibodies in each case, acquired from the following vendors: anti-phosphoglycerate kinase (Pgk1, 459250; Invitrogen), anti-Rpb1 (8WG16, Covance), anti-Rpb3 and Rpb4 (Biolegend), anti-CTD-Ser2P (ab5095, Abcam), anti-CTD-Ser5P (clone 4H8, Millipore), and anti-Thr4P (clone 6D7, Active Motif).

Techniques: Mutagenesis, SDS Page, Western Blot, Quantitation Assay, Co-Immunoprecipitation Assay, Immunoprecipitation