Supersignal west maximum sensitivity substrate

Cells were lysed with RIPA buffer (Santa Cruz, sc-24948) containing protease inhibitors (Cocktail, Roche). Protein lysate concentration was quantified using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA). Protein was resolved on denatured SDS-PAGE. The separated proteins were transferred to nitrocellulose membrane. The membrane was blocked in 3% BSA at room temperature (RT) for 1 hr, incubated with primary antibodies overnight at 4℃, washed 3 times for 5 min, and incubated with secondary antibody for 1 hr at RT. The membrane was then washed and developed using SuperSignal West Maximum Sensitivity Substrate (Thermo Fisher Scientific).

Protein samples were resolved on 10% Criterion Tris-HCl precast gels (Biorad, #3450011) or in the case of the phosphorylated species of Arpp19 and ENSA on a 10% Tris-HCl SDS-PAGE supplemented with 10 μM Phos-tag reagent (NARD Institute, AAL-107) and then transferred onto PVDF membranes. The following antibodies were used for detection of the respective proteins: mouse α-Cdc27 (BD Biosciences, #610455), mouse α-cyclin B2 (Santa Cruz Biotechnology, #sc-53239), rabbit α-Nup53 serum,^{85 (link)} rabbit α-PPP1A pT320 (Abcam, #ab62334), rabbit α-Cdc25C,^{44 (link)} rabbit α-Wee1 pT150,^{86 (link)} rabbit α-Greatwall serum,^{60 (link)} rabbit α-ENSA serum,^{60 (link)} rabbit α-Arpp19,^{63 (link)} rabbit α-ENSA pS67/Arpp19 pS62 (Cell Signaling Technology, #5240) and rabbit α-Cdk1 pY15 (Cell Signaling Technology, #9111L). For the detection of the primary antibody, peroxidase-linked sheep α-mouse IgG (GE Healthcare, #NA931V) and AMDEX goat α-rabbit IgG (GE Healthcare, RPN4301) was used. Chemiluminescence was detected on a BioRad ChemiDoc MP Imaging system using SuperSignal West Maximum Sensitivity Substrate (ThermoFisher Scientific, #34095) or Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, #WBLKS0500).