mouse anti dna rna hybrid antibody  (ATCC)

Journal: bioRxiv

Article Title: The 7SK small nuclear ribonucleoprotein links the cell responses to transcription and replication stress by promoting replication fork reversal and homologous recombination

doi: 10.1101/2025.09.30.678750

Figure Lengend Snippet: A) Schematic of active P-TEFb release from 7SK-snRNP to promote RNA polymerase II (RNAPII) pause release in response to stress and growth stimuli. B) Protein levels of HEXIM1, LARP7 and Tubulin (loading control) in U2OS cells 48 h after siRNA transfection. nonT = non-targeting control siRNA. C) Approach for siRNA depletion or small molecule inhibitor treatment prior to DNA fibre labelling in presence of camptothecin (CPT, 10 μM) or hydroxyurea (HU, 200 μM). D) Replication fork speeds (IdU label) after CPT or HU treatment in presence of HEXIM1 (+) or nonT (-) siRNA. Data from 2 repeats. E) Replication fork speeds (IdU label) after CPT or HU treatment in presence of LARP7 (+) or nonT (-) siRNA. Data from 2-3 repeats. F) DRB inhibits P-TEFb and p38 MAPK inhibitor (P38i) inhibits 7SK-snRNP/P-TEFb dissociation. G) Replication fork speeds (IdU label) after CPT or HU treatment +/- p38i. Data from 3 repeats. H) Replication fork speeds (IdU label) after CPT or HU treatment +/- 100 μM DRB. Data from 2 repeats. I) Nascent chromatin capture proteomics in HeLa S3 cells shows limited enrichment or depletion of HEXIM1, LARP7 and MEPCE at ongoing (nascent/mature chromatin), stalled (3 mM HU, 30 min/untreated) or collapsed (1 μM CPT, 40 min + roscovinite/roscovitine) replication forks compared to PCNA or RAD51. Roscovitine was included to prevent new origin firing. Scatter graphs show aggregates and medians (black points) of independent repeats with overall median (line). Kruskal-Wallis with multiple comparisons test, **** p ≤ 0.0001; ns: not significant.

Article Snippet: The membrane was then incubated overnight at 4 °C in mouse anti-DNA-RNA hybrid antibody (S9.6 hybridoma growth medium, ATCC HB-8730, 1:1,000) or mouse anti-dsDNA antibody (Abcam ab27156, 1:100,000) in sterile 5 % BSA/TBST, before being washed in TBST and incubated in goat anti-mouse HRP (Cell Signaling 7074, 1:5,000) 5 % milk/TBST for 1 hour at room temperature.

Techniques: Control, Transfection

Journal: bioRxiv

Article Title: The 7SK small nuclear ribonucleoprotein links the cell responses to transcription and replication stress by promoting replication fork reversal and homologous recombination

doi: 10.1101/2025.09.30.678750

Figure Lengend Snippet: A) Protein levels of CDK9 in soluble extract (SE) and nuclear pellet (NP) fractions after 2 h treatment with 10 μM CPT or 200 μM HU +/- HEXIM1 siRNA (48 h). B) Protein levels of CDK9 in SE and nuclear NP fractions +/- LARP7 siRNA as in A. C) Relative levels of SE (7SK-snRNP bound) versus NP (chromatin-bound) P-TEFb after treatment +/- HEXIM1 siRNA as in A. n=3. D) Relative levels of SE versus NP P-TEFb after treatment +/- LARP7 siRNA as in B. n=3. E) Representative images of nascent RNA labelling with 5-ethynyluridine (EU). DNA was detected using DAPI. Bars: 50 μm. F) Nuclear EU intensities after treatment with 10 μM CPT and EU for 20 or 50 min. n=1. G) Nuclear EU intensities after treatment with 10 μM CPT or 200 μM HU (2 h) +/- HEXIM1 siRNA. n=3. H) Slot blots of genomic DNA stained with S9.6 antibody (RNA:DNA hybrids) and double-stranded DNA (dsDNA; loading control) after treatment with 10 μM CPT or 200 μM HU (20 min) +/- HEXIM1 siRNA. RNase H treatment was used as control. I) RNA:DNA hybrid quantification as in E. n=4. J) Replication fork speeds (IdU label) after CPT or HU treatment overexpression of turboGFP-RNase H1 (+) or eGFP vector control (-). Data from 2 repeats. K) Approach for siRNA depletion and 10 μM CPT or 200 μM HU treatment prior to immunostaining. L) Percentages of cells with >10 γH2AX foci after 2 h CPT or HU +/- HEXIM1 siRNA. n=2-3. M) Percentages of cells with >9 53BP1 foci after 2 h CPT or HU +/- HEXIM1 siRNA. n=3. Scatter blots show aggregates and medians (black points) of independent repeats with overall median (line). ANOVA or Kruskal-Wallis with multiple comparisons test, * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns: not significant.

Article Snippet: The membrane was then incubated overnight at 4 °C in mouse anti-DNA-RNA hybrid antibody (S9.6 hybridoma growth medium, ATCC HB-8730, 1:1,000) or mouse anti-dsDNA antibody (Abcam ab27156, 1:100,000) in sterile 5 % BSA/TBST, before being washed in TBST and incubated in goat anti-mouse HRP (Cell Signaling 7074, 1:5,000) 5 % milk/TBST for 1 hour at room temperature.

Techniques: Staining, Control, Over Expression, Plasmid Preparation, Immunostaining

Journal: BMC Methods

Article Title: Quantitative single-molecule FLIM and PIE-FRET imaging of biomolecular systems

doi: 10.1186/s44330-025-00048-1

Figure Lengend Snippet: A Annealed RNA/DNA hybrid MFU substrate was diluted to 15 pM and single molecules were attached to a homebuilt flow cell via biotin-streptavidin linkage, followed by flushing the channel with Image Buffer C (Materials and Methods). An RNA(pink, 15 bases)/DNA(green, 45 bases) hybrid oligo and a complementary DNA strand (green, 45 bases) were all purchased from IDT. The complementary DNA strand was labeled with a donor (Atto550, green circle), and the RNA segment of the hybrid is labeled with an acceptor (Atto647N, red circle). The DNA acceptor strand is biotinylated at the 5’ end for attachment via biotin-streptavidin linkage. At least 20 substrates were probed, but we present detailed analysis for a single trace. We clicked on a single biomolecule (white circle) and collected photons from that location to produce fluorescence intensity traces for the donor (blue, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{D}_{ex}{D}_{em}$$\end{document} ) and acceptor (magenta, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{A}_{ex}{A}_{em}$$\end{document} ) fluorophores attached to the biomolecule. The green trace is the FRET \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{(D}_{ex}{A}_{em}$$\end{document} ) signal, and the calculated FRET efficiency is below in black. We binned the photon arrival macrotimes for each signal in 100 ms bins. The calculated FRET efficiency below (black scatter, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\: \langle E\, \rangle =0.61$$\end{document} ) was corrected for leakage and direct excitation as described in the text. We interpret the loss of the acceptor signal as photobleaching since there is no Nsp15 in this experiment. B Fluorescently labeled Thermus aquaticus MutL enzymes were encapsulated in liposomes and attached to the surface of a homebuilt flow cell. C The liposomes were imaged in PIE mode, and the images were separated based on the excitation laser time gate and detection channel. A fast pattern matching algorithm that separates the photons based on excitation laser time gate and detection channel creates a single RGB image with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{D}_{ex}{D}_{em}$$\end{document} in green, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{A}_{ex}{A}_{em}$$\end{document} in red. Some proteins are donor-only or acceptor-only labeled. Double-labeled (donor-acceptor) may be identified with a yellow color if the photon counts are high enough. D The \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{D}_{ex}{D}_{em}$$\end{document} , \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{D}_{ex}{A}_{em}$$\end{document} (FRET), and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{A}_{ex}{A}_{em}$$\end{document} images obtained simultaneously. E Three representative traces for different encapsulated MutL enzymes. We clicked on a single encapsulated enzyme (For trace 1, indicated by the white circle in C and D) and collected photons from a single location to produce fluorescence intensity traces for the donor (blue) and acceptor (magenta) attached to the biomolecule. The green trace is the FRET \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{(D}_{ex}{A}_{em}$$\end{document} ) signal, and the calculated FRET efficiency is below in black. The calculated FRET efficiency was corrected for leakage and direct excitation as described in the text. The calculated gamma factor is indicated on each trace, and a histogram of the calculated corrected FRET efficiency is shown below each trace (gray bars)

Article Snippet: The donor-labeled (Atto550) DNA strand was annealed to the acceptor-labeled (Atto647N) hybrid RNA/DNA strand by heating equimolar amounts of both strands in annealing buffer (10 mM Tris pH 7.5, 50 mM NaCl, 1 mM EDTA) to 95 °C for 5 min in a Bio-Rad T100 thermal cycler, followed by cooling to 25 °C over 70 min at −1 °C per minute, and then stored at 4 °C until further use.

Techniques: Labeling, Fluorescence, Liposomes