Anti cyclin a antibody

HeLa cells were cultured on coverslips (Matsunami Glass, Osaka, Japan) and irradiated using an MX-80Labo (mediXtec) at a dose of 5 or 10 Gy. After 4, 6, and 24 h, the irradiated cells were harvested and fixed with 4% paraformaldehyde. After permeabilization with 0.3% Triton X-100 in PBS, the cells were incubated with anti-RAD51 antibody (dilution 1:500–1000 H-92; Santa Cruz Biotechnology). Anti-cyclin A antibody (dilution 1:1000 B8; Santa Cruz Biotechnology) was used to identify the S to G2 cell cycle phases. Alexa Fluor-568-conjugated goat anti-rabbit IgG (dilution 1:1000; Thermo Fisher Scientific) and Alexa Fluor 488 goat anti-mouse IgG (dilution 1:1000, Thermo Fisher Scientific) were used as secondary antibodies. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; dilution 1:10,000, Thermo Fisher Scientific); RAD51 foci were examined under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Cells with more than five RAD51 foci were considered RAD51 focus-positive cells. The RAD51 foci were examined under a confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany) with ZEISS ZEN Software (Carl Zeiss AG). The number of RAD51 foci present in the nuclei stained with DAPI and the signal of cyclin A were quantified.

The HeLa, HepG2, and HEK293T cell lines were obtained from RIKEN Cell Bank and grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The following antibodies were used for Western blotting and immunostaining: anti-RAD51 (dilution 1:500–1000 H-92; Santa Cruz Biotechnology, Dallas, TX, USA), anti-BRCA2 (dilution 1:250 OP-95; Merck, Darmstadt, Germany), anti-lamin B1 (dilution 1:1000 PM064; MBL, Nagoya, Japan), anti-FLAG (dilution 1:1000 M2; Merck), anti-α-Tubulin (dilution 1:1000 M175-3; MBL), anti-GFP (dilution 1:1000 598; MBL), and anti-Cyclin A antibody (dilution 1:1000 B8; Santa Cruz Biotechnology).
The FLAG-HA- or FLAG-HA-EGFP-fused BRCA2 C-terminus (3260–3418 aa) and FLAG-HA-fused CTRBD (3260–3331 aa) were stably expressed using the pOZ-N plasmid [28 (link)]. The shRNA-mediated knockdown of BRCA2 was achieved by expressing the target sequence 5′-TACAATGTACACATGTAACAC-3′ in the pLKO.1 vector (donated by David Root; Addgene plasmid no. 10878) and scramble shRNA (donated by David Sabatini; Addgene plasmid no. 1864) [29 (link),30 (link)]. The plasmid DNA was transfected into HEK293T cells using FuGENE HD (Promega, Madison, WI, USA) in accordance with the manufacturer’s instructions.