Rabbit anti γh2a x phospho s139

PK-15 cells (1–5×10⁶) were incubated with 500 ng/ml of recombinant proteins for 12 h in 12-well tissue culture plates (Nest, China), washed 3 times with PBS, cells fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 0.2% Triton X-100 for 15 min. After blocking with 3% FBS in PBS for 1 h, the cells were incubated with rabbit anti-γH2A.X (phospho S139) (Abcam) overnight at 4°C. washed 5 times with PBST, and incubated with anti-rabbit IgG (H + L)-FITC antibody produced in goat (Sigma) at 37°C for 1 h. Nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI) (Beyotime) for 15 min and the γ-H2A.X foci examined in a Leica SP2 Confocal system (Leica Microsystems, Germany).

Unless otherwise specified, all chemicals and reagents were of analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Amresco (Solon, OH, USA). NR was a kind gift from Prof. Marie Migaud (Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA). Cell culture reagents and lab plasticware were from Gibco (Waltham, MA, USA), Greiner Bio-One (Monroe, NC, USA), and Orange Scientific (Braine-l’Alleud, Belgium). The ultrapure water was obtained from a Milli-Q Synthesis purification system (Millipore, Burlington, MA, USA). The following antibodies were used: rabbit anti-γH2AX (phospho S139) (abcam, Cambridge, UK, ab81299), mouse monoclonal anti-Ki-67 (Thermo Fisher Sci., Waltham, MA, USA, MA1-2020), mouse monoclonal anti-phospho-ATM (S1981) (abcam, Cambridge, UK, ab36810), secondary antibodies: Alexa Fluor 488-conjugated goat anti-rabbit IgG, Alexa Fluor 568-conjugated goat anti-mouse IgG, Alexa Fluor 568-conjugated goat anti-rabbit IgG, and Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR, USA, A11008, A11004, A11011, A11001). Click-iT Plus EdU imaging kit was obtained from Invitrogen (Waltham, MA, USA, C10086).

HEK293T cells were cultured on glass slides in a six-well dish. After SpCas9/Cas9TX transfection or 10 μM etoposide (Sigma, S1225) treatment for 24 h, the cells on the slides were fixed in 4% PFA for 10 min at RT, followed by washing in PBS and permeabilization with 0.5% TritonX-100 for 15 min. Before primary antibody staining, cells were blocked using 3% FBS for 60 min. The cells were incubated with rabbit anti-γH2A.X (phospho S139) (Abcam, ab2893) at 1:500 dilution for 1 h at room temperature or overnight at 4 °C, followed by washing in 0.2% Tween. Next, the cells were stained with Alexa 488 Fluor goat anti-rabbit IgG HRP (Abcam, ab6721) secondary antibody at a 1:500 dilution for 60 min at RT, and nuclear staining was performed using Hoechst 33342 (Sigma, B2261) at 1 mg/mL for 15 min at RT. Finally, the slides were mounted to microscope slides for microscopy analysis. Images were acquired using a Nikon A1R high-speed laser confocal microscope and analyzed by ImageJ (version 1.51 J8) according to the instructions included with the software.