Raw_data_RPA_RNaseH1

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Raw data of immunoblot and dot-blot

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Immunofluorescence and immunoblotting Immunofluorescence was performed as previously described20. Briefly, cells were fixed with 3% paraformaldehyde and permeabilized with 0.3% Triton X-100. The secondary antibodies were Alexa Fluor 488-, 532- 546- or 633-conjugated anti-mouse and anti-rabbit secondary antibodies (Molecular Probes). For S phase analysis, cells were grown for 3 hours with 50 μM Bromodeoxyuridine (BrdU, Sigma-Aldrich). After fixation, coverslips were treated with HCl (1% and 2%), quenched with borate buffer, and processed for immunofluorescence. 53BP1 (4937 Cell Signaling), BRCA1 (149823 Cell Signaling) and γH2AX (20E3 Cell Signaling) antibodies were used in immunofluorescence. Cells were imaged with a confocal microscope Leica AOBS SP8. Nuclei were stained with Hoechst 33342 (10 μg/ml, Merck). Unless specifically explained, images represent maximum intensity projections of 3D image stacks and were adjusted for brightness and contrast for optimal visualization. Cell lysates after SDS-PAGE and immunoblotting on nitrocellulose (Whatman) were incubated with primary antibodies. HPR-conjugated secondary antibodies were obtained from Cell Signalling and blots were developed with Super Signal West Dura (Thermo Fisher Scientific). Odyssey Infrared Imaging systems (LI-COR Biosciences) was used to detect the fluorescence signal of secondary fluorescent antibodies (anti-Rabbit CF770 SAB4600215, anti-Mouse CF680 SAB4600361 Merck). For stripping, primary and secondary antibodies were removed by using Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific), according to manufacturer. Unless otherwise indicated, all the immunoblot figures were representative of at least two biological replicates. The primary antibodies used in this work are listed in Key resource Table. Dot blot for R-loop quantification gDNA was extracted using the Quick-DNA Miniprep Kit (Zymo Research). The isolated gDNA was treated with 4 units (U) of Ambion™ RNase III (Thermo Fisher Scientific) for 20 minutes at 37°C. Samples were then split in half and control samples were digested with 10 U of RNaseH1 (Thermo Fisher Scientific) for 15 minutes at 37°C. 250 ng of the samples were spotted onto a nitrocellulose membrane (Amersham) using a dot-blot apparatus (Fisher Scientific). DNA was cross-linked to the membrane by UV light, followed by blocking with 1x SSC buffer solution. The membrane was incubated overnight at 4°C with the S9.6 antibody (MABE1095 Merck). After incubation with HRP-conjugated secondary antibodies (Thermo Fisher Scientific), the signal was detected using Super Signal West Dura (Thermo Fisher Scientific). An antibody against dsDNA (MAB1293 Merck) was used as a loading control after stripping the membrane.

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