Antibody Characterization and DNA Damage Assays

Mouse antibody to MASTL (clone 4F9, Millipore MABT372) was described previously (Wang et al., 2011 (link)). Phospho-specific E6AP Ser-218 antibody was generated using a synthesize peptide (SSRIGDS phospho-S QGDNNLQ). Other antibodies include α-tubulin (Santa Cruz Biotechnology, #sc-5286), E6AP (Bethyl Laboratories, A300-351), HA (Cell signaling technology #3724), γ-H2AX Ser-139 (Cell signaling technology #9718S), phospho-ATM/ATR substrate motif (Cell signaling technology, #6966S), phospho-SMC1 Ser-957 (Cell signaling technology, #58052), phospho-CHK1 Ser-345 (Cell signaling technology, #2348), phospho-CHK2 Thr-68 (Cell signaling technology, #2197), Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (Cell signaling technology, #2914), Aurora A (Cell signaling technology, #14475), Aurora B (Cell Signaling technology, #3094), CDK1 (Cell signaling technology, #9112), Cyclin B (Cell signaling technology, #4138), phosphor-CDK substrates (Cell signaling technology, #2325), RPA32 (Thermo Fisher Scientific, # PA5-22256), S5a (Boston Biochem, #SP-400), ubiquitin (Cell Signaling Technology, #3936), and GFP (Cell Signaling Technology, #2555).
The following chemicals were used: Hydroxyurea (HU, MP Biomedicals, #102023), doxorubicin (DOX, MilliporeSigma, #25316-40-9), caffeine (Sigma-Aldrich, #C0750), ATM inhibitor (KU55933, Selleckchem, #S1092), ATR inhibitor (VE-821, Selleckchem, #S8007), cycloheximide (CHX, Fluka analytical, #01810), etoposide (Sigma-Aldrich, #E1383), camptothecin (Sigma-Aldrich, #C9911), G418 sulfate (Thermo Fisher Scientific, #10131035), MG132 (Calbiochem, #133407-82-6), cisplatin (R&D systems, #15663-27-1), propidium iodide (PI, Thermo Fisher Scientific, #P1304MP), and isopropyl-beta-D-thiogalactopyranoside (IPTG, RPI research products international, # 367-93-1).