Anti mdc1

Immunoprecipitation and immunoblotting experiments were performed as described before.¹¹ (link) Briefly, the cells were first transfected with the plasmids or treated with different drugs. Then the cells were collected and washed with PBS, followed by cell lysis in the RIPA buffers.¹¹ (link) The resultant supernatant was collected and the antibodies were added as needed. After incubation overnight at 4°C, Protein A or G Sepharose was added into the solution and incubated further for 1 hour at 4°C. The immunoprecipitates were then subject to extensive wash, then the pellets after centrifugation were collected and boiled in Sample Buffers. The following primary antibodies were used for immunoblotting: anti-MDC1, anti-GFP, anti-UBE3D, anti-PCNA, anti-KAP1, anti-HA, and anti-FLAG M2 (Sigma). Peroxidase-conjugated secondary antibodies were from Jackson Immuno Research. Blotted proteins were visualized using the ECL detection system (Amersham). Signals were detected by a LAS-4000, and analyzed using Multi Gauge (Fujifilm). Micrococcal nuclease assays were carried out as previously described.⁹ (link) All IP experiments were repeated for at least three times.

The full-length cDNA of human OGT was cloned into the pEGFP-C1 vector. Internal deletion mutants and the enzyme-dead mutant (G598S) of OGT were generated using the QuickChange site-directed mutagenesis kit (Stratagene). Deletion mutants of OGT were constructed as described previously (37 (link)).
Antibodies used in this study include the following: anti-OGT antibody (Novaus), anti-γH2AX (Upstate Biotechnology, monoclonal and polyclonal), anti-O-GlcNAc (RL2 monoclonal [Abcam] and CTD110.6 monoclonal [Covance]), anti-FLAG (Sigma), anti-Myc (Sigma), anti-Actin (Sigma), anti-Mdc1 (Sigma), anti-PAR (Genetex, monoclonal), anti-Ku80 (Cell Signaling Technology), anti-Chk1 (Cell Signaling Technology), anti-phospho-Chk1 (Ser345,Cell Signaling Technology) and anti-phospho-SQTQ (Cell Signaling Technology). siRNA sequences were generated to target human OGT (5′-GCACATAGCAATCTGGCTTCC-3′ or 5′-CCAAACTTTCTGGATGCTTAT-3′).
The OGT inhibitor (ST045849) was purchased from TimTec and the O-GlcNAcase (OGA) inhibitor (PUGNAc) was purchased from Toronto Research Chemicals. The ATM inhibitor KU-55933 was purchased from Calbiochem. For cell treatment, a final concentration of 2 μM KU-55933 was added to the cell medium at the indicated timepoints.