Phospho ser thr atm atr substrate

The cells were fixed with 4% paraformaldehyde/PBS (Wako) and permeated through the membrane with 0.5% Triton X-100/tris-buffered saline (TBS) for 1 minutes. The cells were blocked with 1% bovine serum albumin (BSA) and 10% goat serum/TBS for 1 hour at 4 °C. Then, it was made to react with the following antibodies: γH2AX (1: 1,000, 05-636, Millipore, Temecula, CA, USA), phospho-(Ser/Thr) ATM/ATR substrate (1: 500, 2851, Cell Signaling Technology, Danver MA, USA), 53BP1 (1: 250, sc-58749, Santa Cruz). The secondary antibody was reacted using Alexa488 and Alexa594 (Thermo Fisher Scientific), and the nucleus was stained using DAPI (Dojindo, Tokyo, Japan). After immunostaining, DNA damage-positive cells were quantified by observation using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

The cells were fixed with 4% paraformaldehyde/PBS (Fujifilm Wako Chemicals, Tokyo, Japan) for 10 min, and membrane permeation was performed using 0.2% Triton X-100/Tris-buffered saline (TBS) for 5 min at room temperature. Next, the cells were blocked with 1% bovine serum albumin and 10% goat serum in TBS for 1 h at 4 °C, then incubated with the following antibodies in the blocking buffer overnight at 4 °C: γ-H2AX (Merck Millipore, Darmstadt, Germany, #05-636, 1:1000 dilution) and phospho-(Ser/Thr) ATM/ATR substrate (Cell Signaling Technology, Danvers, MA, USA, #2851, 1:500 dilution). The secondary antibodies were Alexa488 anti-rabbit IgG and Alexa594 anti-mouse IgG antibodies (Thermo Fisher Scientific, Waltham, MA, USA, 1:1000 dilution) diluted in blocking buffer, and the cells were incubated for 45 min at room temperature. The nucleus was stained with DAPI (Dojindo, Tokyo, Japan) for 5 min at room temperature. After immunostaining, DNA damage-positive cells were quantified by observation under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).