Bmc9318

The following primary antibodies were used: mouse anti-BrdU antibody clones: B44 and 3D4 (BD Biosciences), Bu20a and MoBu-1 (Exbio Praha), BMC9318 (Roche), Bu5.1 (Millipore), Bu6-4 (GeneTex), B33 (Sigma Aldrich) and chicken polyclonal anti-BrdU antibody (Abcam). For the concurrent localisation of DNA synthesis and cellular proteins, we used following antibodies: mouse anti-SC35 antibody (Abcam), rabbit anti-H1.2 antibody (Abcam), rabbit anti-PCNA antibody (proliferating cell nuclear antigen; Abcam), mouse anti-coilin antibody (Abcam), mouse anti-MBD4 antibody (Santa Cruz Biotechnology), rabbit anti-fibrillarin antibody (Abcam) and mouse anti-mitochondrial antibody (MTC02; Abcam).
The following secondary antibodies were used: DyLight 649 anti-mouse, Alexa Fluor^® 488 anti-mouse, Alexa Fluor^® 488 anti-rabbit and DyLight 649 anti-chicken antibodies (all Jackson ImmunoResearch).

HAC#21-HeLa cells were treated with 2 mM thymidine for 16 hrs, released into normal medium for 11 hrs, and then treated with 1 µg/ml aphidicolin for 14 hrs. Following release, cells were pulse-labeled with 50 µM 5-bromo-2′-deoxyuridine (BrdU) for one-hour intervals, chased with normal medium, and harvested 9 hrs post release. For monitoring cell cycle progression, a parallel sample of cells was harvested at each time point, stained with propidium iodide, and analyzed for DNA content by FACS Calibur (BD). For replication analysis, genomic DNA, extracted using a Wizard Genomic DNA Extraction kit (Promega), was sonicated to a few hundred bp using a Bioruptor (BioRad). 2 µg of the sonicated DNA and 2 ng of BrdU-substituted/sonicated Escherichia coli DNA were heat-denatured in a mixture and immunoprecipitated with 5 µg of anti-BrdU antibody (Roche, BMC9318) in 100 µl of IP buffer (0.05% Triton X-100, 140 mM NaCl, and 10 mM sodium phosphate, pH 7.2) for 30 min at room temperature (RT), and further rotated for 30 min with 20 µl of Dynal M-280 sheep anti-mouse IgG (Invitrogen). The beads were washed four times each for 5 min with 500 µl of IP buffer, eluted twice in 100 µl of 1% SDS/TE at 100°C for 3 min, and DNA was de-proteinized, purified, and subjected to real-time PCR as in ChIP. The graphs and regression curves were drawn with KaleidaGraph (Hulinks).

The following monoclonal anti-bromodeoxyuridine antibody clones were tested in this study: BMC9318 (Roche), Bu-33 (Sigma Aldrich), B44 (Becton Dickinson), Bu6-4 (Genetex), Bu20a (BioLegend) and Bu5.1 (Millipore). As a secondary antibody, we used anti-mouse antibody conjugated with the fluorochrome Alexa Fluor 488 (1:100, Jackson ImmunoResearch).

Verification of synchronization and determination of drug effects was performed by measuring the incorporation of bromo-deoxyuridine (BrdU) into replicating foci within nuclei. At times indicated, cells were pulse-labeled with 15 µM BrdU for 30 min, fixed with 4% paraformaldehyde, and analyzed by standard immunofluorescent techniques^{36 (link), 50 (link)} with an anti-BrdU monoclonal antibody (Roche, clone no. BMC9318, RRID:AB_2313622). The average counts of three fields of 100 or more cells were used to determine the percentages of BrdU-labeled nuclei, +/− 1 s.d.

Paraffin-embedded tissue was sectioned freshly (4.5 μm) and dried overnight at 37 °C. Slides were deparaffinized with xylene and rehydrated in a graded series of ethanol. Endogenous peroxidase activity was blocked with 3% H₂O₂ in methanol. For antigen retrieval, tissue was cooked in 0.01 M sodium citrate solution (pH 6.0) for 20 min. Non-specific binding was prevented by incubation with PBT (phosphate-buffered saline, bovine serum albumin 10 mg/ml, and Triton X-100 0.1%). Tissue sections were incubated overnight with a primary antibody.
Mayer’s hematoxylin (Sigma-Aldrich) was used as nuclear counter stain. The following antibodies were used: anti-mmASS1 (1:2000) [35 (link)], anti-hsASS1 (1:2000, clone C104588; Sigma, Deisenhofen, Germany), and anti-BrdU (1:500, clone BMC9318; Roche, Woerden, The Netherlands). Images were captured with an Olympus BX51 microscope.