Anti rad51

Laser scanning confocal microscopy was performed to detect RAD51 and γH2AX foci in cancer cells. Briefly, ovarian cancer cell, CP70 were grown on coverslips and treated with 10 µM etoposide or vehicle for 24 h followed by an additional 24 h in drug-free media. Coverslips were washed with PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS. Cells were permeabilized with 0.01% triton-X 100 (Fisher Scientific) for 5 min followed by a wash with chilled PBS and blocked with 3% goat serum (Thermo Scientific) for 1 h at room temperature. Cells were incubated with anti-RAD51 (1:250, Abcam) or anti-γH2AX (1:300, Cell Signaling Technology) antibodies overnight at 4 °C in a humidified chamber. Next, cover slips were washed 3X with PBS. Alexa fluorescent conjugated secondary antibodies were added to the coverslips and incubated for 1 h. Coverslips were washed 3 × and mounted with DAPI containing Vectashield (Vector Lab). Images were captured by confocal microscope at 63 × magnification in oil emersion (Leica SP8 confocal microscope). RAD51 and γH2AX foci were counted on 100 representative cells by image J software.

HR was assessed as previously described [25 (link)]. Cycling cells were seeded at 40,000 cells/cm² and incubated for 24 h. Cells were exposed to 200 mJ/cm² of UV-C radiation, incubated for 2 h, and fixed in ice-cold methanol. Cells were permeabilised and treated with 1 µg/ml anti-phosphorylated H2AX (Millipore, 05-636-I) and 1:1000 anti-Rad51 (Abcam, ab133534) antibodies followed by exposure to 2 ug/ml Alexa Fluor 546 and 488 antibodies (Invitrogen, A-11003 and A-11008). Cells were imaged with a Zeiss Axio Observer Z1 and Zen 2.3 software. ImageJ software identified DAPI nuclear regions. A twofold increase in the average number of phosphorylated H2AX foci in irradiated versus control cells demonstrated sufficient cellular assault. Our previously validated threshold of a twofold increase in average Rad51 foci number versus control signalled a competent cell [25 (link)]. Antibodies are provided in Supplementary Information Section 1.2.

Proteins were extracted after cells were exposed to cisplatin (IC₅₀) for 48 h using ice-cold RIPA buffer (Beyotime, Chongqing, China) supplemented with phenylmethanesulfonyl fluoride (PMSF); the concentration was determined with a BCA kit (Beyotime). Proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, Billerica, MA, United States). Primary antibodies were as follows: anti-NTNG1 (GeneTex), anti-RAD51 (Abcam, Cambridge, United Kingdom), anti-AXL/p-AXL (Cell Signaling Technology, Danvers, MA, United States), anti-Akt/p-Akt (Cell Signaling Technol.), anti-GAS6 (Bioss Biotechnology, Beijing, China), and anti-β-actin (Proteintech, Wuhan, China). The secondary antibody was a goat anti-rabbit IgG antibody (Abcam). Bands were analyzed with the software Image Lab (Bio-Rad Lab., Hercules, CA, United States). The density ratio was used to calibrate the level of a target protein, with β-actin as the reference.
To detect the expression level of NTNG1 after cisplatin exposure, proteins were extracted after SKOV3 or SKOV3/DDP cells were exposed to cisplatin (IC₅₀ or 0.5 × IC₅₀) for 48 h, or after SKOV3/DDP cells were cultured in cisplatin-free medium for 3, 5, 7, and 9 days.