Anti phospho chk2 thr68

Calcium- and magnesium-free phosphate-buffered saline PBS(−) and paclitaxel (#169-18 611) were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Propidium iodide (PI) and the chk2 inhibitor II were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). SCH900776 was purchased from Med Chem Express (Shanghai, China). The anti-rabbit horseradish peroxidase (HRP)-conjugated IgG (#7074), and the anti-mouse HRP-conjugated IgG (#7076) secondary antibodies, as well as the anti-phospho-cdc2 (Tyr15) (#9111), the anti-cyclin B1 (#4135), the anti-phospho-histone H3 (Ser10) XP (#3377), the anti-phospho-chk1 (Ser296) (#2349), the anti-chk1 (#2360), the anti-phospho-chk2 (Thr68) (#2661), the anti-p21 (#2947) and the anti-β-actin (#4967) monoclonal antibodies were purchased from Cell Signaling Technology Inc (Danvers, MA, USA). The anti-DAP3 (Cat. No. 610662) monoclonal primary antibody was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The Ambion Silencer® Select Pre-designed siRNA against the gene-encoding DAP3 (Cat. No. s1506) and the Silencer® Select Negative #1 Control (Cat. No. AM4611) siRNAs were purchased from Thermo Fisher Scientific, Inc (Waltham, MA, USA).

Whole cell lysate were immunoblotted as previously described [5 (link)]. The primary antibodies used for immunoblotting were described as follows. The rabbit antibody against human BMCC1 was generated by MBL (Nagoya, Japan) [5 (link)]. The Anti-E2F1 (#3742), anti-phospho-ATM (Ser1981) (#4526), anti-phospho-Chk2 (Thr68) (#2661), anti-phospho-H2A.X (Ser139) (#9718), anti-phospho-FOXO3a (Thr32) (#9464), anti-FOXO3a (#2497), anti-PARP (#9542, Fig. 5a and b) and anti-caspase-9 (#9502), anti-Bim (#4582), HRP-conjugated anti-Rabbit (#7074) and anti-Mouse (#7076) secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-Human phospho-E2F1 (pS364) was purchased from ROCKLAND (Limerick, PA) and anti-p73 (ab-3) was obtained from Millipore (Billerica, MA), whereas anti-ATM (sc-23,921), anti-PARP-1 (sc-8007), and anti-Actin (sc-8432) were bought from Santa Cruz Biotechnology (Dallas, TX).

Immunoblotting was performed as previously described [42 (link)]. The following commercially available antibodies were used: anti-p21 (C-19; Santa Cruz Biotechnology, Santa Cruz, CA, USA), horseradish peroxidase-conjugated anti-p53 antibody (DO-1; Santa Cruz Biotechnology), anti-p53 (DO-1; EMD Millipore, Darmstadt, Germany), anti-p27 (Clone 57/Kip1/p27; BD Bioscience, Franklin Lakes, NJ, USA), anti-acetyl-p53 (Lys382) (#2525; Cell Signaling Technology, Beverly, MA, USA), anti-phospho-p53 (Ser15) (#9284; Cell Signaling Technology), anti-phospho-Chk2 (Thr68) (#2661; Cell Signaling Technology), anti-Chk2 (#2662; Cell Signaling Technology), anti-phospho-Chk1 (Ser345) (#2348; Cell Signaling Technology), anti-Chk1 (#2360; Cell Signaling Technology), anti-ATM (#2873; Cell Signaling Technology), anti-phospho-histone H2AX (Ser139) (#9718; Cell Signaling Technology), and anti-β-actin (2F3) (Wako, Osaka, Japan).

For Western blotting, HeLa cells were lysed with RIPA buffer (see Section 2.4), and 50 μg of protein was mixed with Laemmli sample buffer [13 (link)] and resolved in 12% SDS-PAGE gels. Proteins were transferred to nitrocellulose membrane (Millipore, Billerica, MA, USA), and membranes were blocked in TBS-T with 5% milk, for 1 h at room temperature. Then, membranes were incubated with one of the following primary antibodies diluted in TBS-T: anti-phospho-Chk1 Ser345 (Cat. number 2341), anti-phospho-Chk2 Thr-68 (Cat. number 2661), or anti-phospho-H2AX Ser139 (Cat. number 9718) polyclonal/monoclonal antibodies from Cell Signaling (Danvers, MA, USA) or an anti-α-Tubulin polyclonal/monoclonal antibody (B-7, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were incubated with appropriate species-specific IRDye (Infrared Dye) secondary antibodies (680 or 800 nm, diluted to 1 : 15000 in TBS-T) for 1 h and visualized and analyzed (by band density quantification) using an Odyssey Infrared Imaging System and the Odyssey V3.0 software (both from Li-COR Biosciences, Lincoln, NE, USA).

Immunoblotting was performed as described previously [31 (link)]. Anti-phospho-Chk2 Thr-68, and phospho-ATM Ser-1981 antibodies were purchased from Cell Signaling (Danvers, MA); anti-phospho-H3 Ser-10, active caspase-3, β-Actin antibodies were obtained from Abcam (Cambridge, MA). Monoclonal antibody to Mastl (clone 4F9, Millipore) was generated against the C-terminus of Mastl. The intensity of band signals was measured using NIH Image-J software.

Cells were grown on coverslips, and fixed with methanol after the indicated treatments. Following blocking with 3% BSA for 1 h, slides were incubated with the primary antibodies at 4 °C for overnight, washed three times, and incubated with the secondary antibodies (Alexa Fluor 488 goat anti-mouse IgG or Alexa Fluor 594 donkey anti-rabbit IgG, 1:700 dilution) at room temperature for 1 h. Slides were mounted on Vectashild-DAPI mounting medium (Vector Laboratories). Primary antibodies used for immunofluorescence studies were anti-Ape1 (Santa Cruz, sc-17774, 1:200), anti-phospho-Chk2 (Thr68) (Cell Signaling, #2661, 1:200), anti-BRCA1 (Calbiochem, MS110, 1:100), anti-γH2AX (Cell Signaling, #9718, 1:500), anti-53BP1 (Cell Signaling, #4937, 1:100), and anti-Rad51 (Abcam, ab213, 1:200). Images were taken by confocal laser scanning microscopy (LSM780, Zeiss), and analyzed by ZEN software (Zeiss).