Anti ph2ax ser139

Total proteins were isolated from the cell extracts, and 30 μg of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After probing with primary antibodies, membranes were washed in Tris-buffered saline that contained 0.05% Tween-20 (TBST) and incubated with a horse-radish peroxidase-linked secondary antibody. Finally, the obtained bands were detected using an Enhanced Chemiluminescence Detection Kit (Amersham). The primary antibodies used were as follows: anti-FLAG (Sigma-Aldrich), anti-p-H2AX (Ser-139) (Cell Signaling Technology), anti-ROC1 (Epitomics), anti-DDB1 (Epitomics), anti-DCAF1 (ProteinTech), anti-RASSF5 (Abcam), anti-TET1 (Abcam) and anti-ERK1/2 (Santa Cruz Biotechnology) antibodies (1:1000).

Western blotting was performed according to standard procedures. We used the following primary antibodies: anti-HOOK1 (Abcam, ab151756), anti-CASP3 (Cell Signaling, #9664), anti-CASP9 (Cell Signaling, #9502), anti-PARP (Cell Signaling, #9532), anti-p-H2AX (Ser139) (Cell Signaling, #9718), anti-NANOG (Santa Cruz Biotechnology, sc-293,121), anti-OCT3/4 (Santa Cruz Biotechnology, sc-5279), anti-KLF4 (Abcam, ab72543), anti-ATF6α (Santa Cruz Biotechnology, sc-166,659), anti-ATF4 (Santa Cruz Biotechnology, sc-390,063), anti-GRP78 (Santa Cruz Biotechnology, sc-13,539), anti-CHOP (Cell Signaling, #2895), anti-LC3B (Abcam, ab48394), anti-p62 (Abcam, ab109012), and anti-B-actin (Abcam, ab16039) as a loading control. We used the following secondary antibodies: rabbit anti-mouse (Abcam, ab97046) and goat anti-rabbit (Abcam, ab97051). The proteins were detected using an ECL detection system (Amersham Biosciences) and a Bio-Rad Chemidoc Touch.

BC cells were seeded on glass coverslips and cultured for 16 h. Then, 10 nM doxorubicin, 100 μM olaparib, or 10 nM doxorubicin plus 30 μM olaparib for combined treatment was added. After 24 h, the coverslips were fixed in 4% paraformaldehyde for 5 min at room temperature, washed twice with PBS, permeabilized with 0.5% Triton X-100 in PBS for 5 min and washed twice with PBS. Samples were incubated in blocking solution (PBS plus 3% bovine serum albumin) at 37 °C for 1 h, followed by incubation for 2 h at room temperature with an anti-53BP1 antibody (Novus Biologicals, NB100-304) diluted 1:100 or anti-pH2AX (Ser139) (Cell Signaling, 9718 S) diluted 1:400. After washing with PBS, cells were incubated with a species-specific Alexa 488-conjugated secondary antibody diluted 1:500 in blocking buffer for 1 h at room temperature in the dark. The nuclei were counterstained with DAPI, and the slides were mounted using Prolong Gold Antifade reagent (Life Technologies). The samples were visualized under an inverted System microscope (Olympus IX-71-ZDC). The mean fluorescence intensity was measured for a minimum of 300 cells per condition using Olympus imaging software. The plotted values represent the means of each condition. Furthermore, nuclear size was determined, measuring at least 50 cells, using ImageJ software.