Rabbit anti cyclin a2

Cells were harvested, washed with PBS, and lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 0.1% SDS, 1% NP-40, and 1% Triton X-100) supplemented with 1 mM PMSF (Sigma) and a protease inhibitor cocktail (Roche) at 4°C for 20 min. The lysates were then centrifuged for 15 min at 12,000 rpm at 4°C. The supernatants were collected, and an equal volume of 2X Laemmli’s buffer was added. The sample was boiled for 5 min at 95°C. Proteins were resolved by 10 or 12.5% SDS-PAGE and then transferred to nitrocellulose membranes (Pall Corporation). Membranes were blocked with 5% non-fat milk in TBST (0.1% Tween 20) for 1 h before incubation with primary and secondary antibodies sequentially. Signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions. The following antibodies were used: rabbit anti-FOP (Abcam, ab156013, 1:2,000), mouse anti-GFP (Santa Cruz, sc-9996, 1:5,000), rabbit anti-AURKA (Cell signaling Technology, 14475, 1:2,000), rabbit anti-Cyclin A2 (Abcam, ab18159, 1:10,000), rabbit anti-pCDC2 (Tyr15) (Cell Signaling Technology, 9111, 1:2,000), rabbit anti-pRb (Ser807/811) (Cell Signaling Technology, 8516, 1:2,000), and mouse anti-β-actin (Sigma, A5441, 1:5,000).

Neuronal cells were harvested with mild trypsinization and immediately fixed with 4% paraformaldehyde for 30 min at 4°C. Neurons were permeabilized with 0.1% Triton-X100 solution in phosphate-buffered saline (PBS) for 10 min on ice and blocked with 3% bovine serum albumin (BSA) solution in PBS for 30 min. Neurons were then processed for immunostaining by 2 hr incubation at 4°C with rabbit anti-Cyclin A2 (1:300; Abcam, Cambridge, UK) or rabbit anti-ChK1 (phospho S317) (1:300; Abcam, Cambridge, UK), followed by incubation for 1 h at RT with Alexa-Fluor 488-conjugated anti-rabbit secondary antibody (1:300; Invitrogen, Thermo Fisher Scientific). Positive neurons were scored either on a Coulter FC500 flow cytometer or on a CyFlow ML flow cytometer (Partec, Canterbury, Kent, UK).