Phh3s10

For immunohistochemistry analysis, representative sections were deparaffinized, rehydrated in graded alcohols, and subjected to antigen retrieval by microwave oven treatment using standard procedures. H & E staining was carried out using Gill's hematoxylin (Poly Scientific R & D Corp.) for 10 min as per the manufacturer's protocol followed by counterstaining with eosin (Poly Scientific R & D Corp.) for 4 min. The immunohistochemistry was performed at Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using Discovery XT processor (Ventana Medical Systems). For PHH3S10 (5 mg/mL, Cell Signaling Technologies INC.) and cleaved caspase 3 (Asp175) (0.1 mg/mL, Cell Signaling Technologies INC.) were used followed by biotinylated goat anti-rabbit IgG Streptavidin-HRP and DAB detection kit (Ventana Medical Systems) according to the manufacturer instructions.

Antibodies against Chk1, pChk1 (S317), pChk1 (S345), pChk2 (T68), pChk2 (S516), γH2AX, pCdc2 (Y15), pCdc25c (S216), Cdc25a, phH3 (S10), PARP, cleaved PARP, 53BP1, cyclin A, cyclin B1, cyclin D, cyclinE, pCDK2 (T160) and RPA70 were purchased from Cell Signaling Technologies and pChk1 (S296) from Abcam.
Cells were washed once with PBS and lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche). Protein concentration was determined using a BCA kit (Pierce). Equal amounts of lysate were separated by SDS-PAGE and western blot analysis conducted using the antibodies indicated above

Antibodies against Cdc25C were from Cell Signaling Technologies (#4688S, Danvers, MA, USA) and Santa Cruz Biotechnology (#SC-327, Dallas, TX, USA). Antibodies against P-Cdc25C (S198), Wee1, P-Cdk1 (Y15), P-MAPK/CDK-substrates (PXS*P and/or S*PXR/K) and P-HH3 (S10) were from Cell Signaling Technologies (#9529S; #13084S; #9111S; #2325S; #9701S respectively). Antibodies against P-Wee1 (Ser53) were from Bioss (#bs-5589R, Woburn, Massachusetts, USA). Antibodies against Cdk1 were from Santa Cruz Biotechnology (#sc-54). Antibodies against MASTL were from Abcam (#ab86387, Cambridge Science Park, Cambridge, UK).