Ab133534

Immunofluorescence staining was performed as described previously (6 (link)). Suspended cells were fixed onto glass using 1% paraformaldehyde and ethanol (70%) at the treatment endpoint. The cells were incubated with primary antibodies against γH2AX (1:500, CST 2577), RAD51 (1:500, Abcam ab133534), RPA (1:200, Abcam ab2175), and p-CHK1 (1:200, Ser317, CST 12302), then incubated with secondary antibody. The cells were visualized using a Zeiss fluorescence microscope.

Proteins were separated with SDS-PAGE and transferred to PVDF membrane. The following antibodies for immunoblotting: BMAL1 (1:2000, NB100-2288, Novus Biologicals), CLOCK (1:2000, NBP1-51610, Novus Biologicals), γH2AX (1:2000, 9718, CST), ATM pS1981 (1:5000, ab81292, Abcam), ATM (1:5000, ab32420, Abcam), RAD51 (1:4000, ab133534, Abcam), ATR (1:5000, ab2905, Abcam), ATR pT1989 (1:1000, GTX128145, GeneTex), RPA1 (1:1000, sc-28304, Santa Cruz), Flag (1:1000, D191041, Sangon Biotech), GAPDH (1:5000, 60004-1-Ig, Proteintech), HRP-conjugated anti-rabbit or anti-mouse (KPL, Inc) were used as secondary antibody.

Fetal oocyte cytospreads were performed as previously described [49 (link)]. Slides were incubated with the first antibody against rabbit/mouse SCP3 (rabbit polyclonal, Novus Biologicals Littleton, NB300-232, USA; or mouse polyclonal, Abcam, ab97672, USA), γH2AX (mouse polyclonal, Abcam, ab26350) or RAD51 (rabbit polyclonal, Abcam, ab133534), for 8 h at 37 °C. After overnight blocking with TBS supplemented with 10% goat serum (Boster, AR009, Wuhan, China), Cy3-labeled goat anti-rabbit (Beyotime, A0516) were used to label SCP3/RAD51 and FITC-conjugated goat anti-mouse antibodies (Beyotime, A0568), were used to label γH2AX/SCP3 at 1:100 dilution for 30 min at 37 °C in the dark; DNA was stained with Hoechst 33342. Pictures were taken with an Olympus fluorescence microscope (BX51).

Cells were treated as indicated. Cells were transfected with siRNA as above in 6-well plates. The next day, cells were seeded in black 96-well plates at 20,000 cells per well for irradiation the following day (48 h post transfection). For ionising radiation, cells were exposed to 10 Gy using an X-ray source and allowed to recover at 37 C for 4 h prior to fixation and imaging. Cells were prepared and stained as previously described [54 (link)], and imaged using a spinning disk confocal microscope. Foci were counted automatically using CellProfiler software. Antibodies used were: RAD51, ab133534, abcam (1:1000); γH2AX, 05-636, Millipore (1:1000).

The cells were cultured on coverslips in 24-well plates. At the indicated time points, the cells were fixed with 4% paraformaldehyde for 10 min and washed with PBS. The fixed cells were permeabilized with 0.1% Triton X-100/PBS for 15 min on ice, incubated with blocking buffer (2% goat serum in 0.3% Triton X-100/PBS) for 10 min, and washed with PBS. The cells were incubated with the primary antibodies mouse anti-γH2AX (#613402, BioLegend, San Diego, CA, USA), rabbit anti-Rad51 (#ab133534, Abcam, Cambridge, UK), rabbit anti-phospho RPA2 (Ser33) (#NB100-544, Novus Biologicals, Littleton, CO, USA), or rabbit anti-53BP1 (#PC712, Merck) in blocking buffer for 60 min. After washing with PBS, the cells were incubated with the corresponding secondary antibodies, including goat anti-mouse IgG (Alexa Fluor 488) (#ab150113, Abcam) or goat anti-rabbit IgG (Alexa Fluor 594) (#ab150080, Abcam) in 0.3% Triton X-100/PBS for 60 min in the dark. The cells were then washed with PBS, and coverslips were mounted on slides with ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific). Immunofluorescence imaging was conducted using a confocal laser microscope (Olympus, FV10i, and FV3000, Shinjuku, Tokyo, Japan).

Cells were seeded on glass coverslips, treated as indicated for each experiment, and then fixed with 4% paraformaldehyde, and permeabilized using 0.1% Triton X-100. Coverslips were incubated with the indicated primary antibodies and then with Alexa Fluor 488 (A-11094, Thermo Fisher Scientific) and 647 (A-32728, Thermo Fisher Scientific) secondary antibodies; nuclei were counterstained using DAPI (D9542, Sigma-Aldrich, Merck). Slides were sealed using ProLong Diamond Antifade Mountant (P36961, Thermo Fisher Scientific). Images were acquired using a Zeiss fluorescence microscope. Primary antibodies: SMYD3 (ab183498, Abcam), 53BP1 (NB100-304, Novus Biologicals), MRE11 (4847, Cell Signaling Technologies), RAD51 (ab133534, Abcam), and RPA32/RPA2 (35869, Cell Signaling Technologies). Densitometric evaluation was performed using ZEN microscopy software. Foci were scored by fluorescence microscopy using a 63 × magnification objective and digital image acquisition on a Zeiss Axio Observer fluorescence microscope.