Antibodies used: Monoclonal ANTI-FLAG, M2 (Sigma, F1804); β-actin (Santa Cruz, sc-47778); Cyclin B1 (Cell Signaling Technology #12231); phospho-p53(Ser15) (Cell Signaling Technology #9286); GFP (Abcam, ab290); CEP68 (Proteintech, 15147-1-AP); Antibodies for phospho-H2AX(Ser139), phospho-H3(Ser10), γ-tubulin, CEP-131, CEP192, Aurora A, SKIV2L2, EFTUD2, eIF4A3, and EXOSC10 are the same ones used for immunofluorescence (above).
Cell pellets were resuspended in RIPA buffer, rotated at 4°C for 30 min, and centrifuged at 13,000 rpm for 20 min. Laemmli buffer was added to the supernatant and protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Blots were blocked in 5% milk or BSA (for phospho-proteins), and incubated in primary antibodies overnight at 4°C at 1:2000 dilutions, except for β-actin (1:7500) and DNAPKcs(S2056P) (1:1000). Blots were then washed 3× in TBST, incubated in HRP-conjugated secondary antibodies (Santa Cruz) (∼1:10000) for 1 h at room temperature, washed 3× in TBST, and detected by enhanced chemiluminescence (BioRad).
Cell pellets were resuspended in RIPA buffer, rotated at 4°C for 30 min, and centrifuged at 13,000 rpm for 20 min. Laemmli buffer was added to the supernatant and protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Blots were blocked in 5% milk or BSA (for phospho-proteins), and incubated in primary antibodies overnight at 4°C at 1:2000 dilutions, except for β-actin (1:7500) and DNAPKcs(S2056P) (1:1000). Blots were then washed 3× in TBST, incubated in HRP-conjugated secondary antibodies (Santa Cruz) (∼1:10000) for 1 h at room temperature, washed 3× in TBST, and detected by enhanced chemiluminescence (BioRad).