Rabbit anti aurka

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).

Cells were lysed in a RIPA lysis and extraction buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitors (Thermo Fisher Scientific) on ice. Samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to nitrocellulose membranes (GE, 10600001). Membranes were probed with primary antibodies against mouse anti-ERK (Cell Signaling, 9107), rabbit anti-phospho-Erk (Cell Signaling, 9101), rabbit anti-IFT88 (Proteintech, 13967-1-AP), mouse anti-ARL6 (Proteintech, 12676-1-AP), rabbit anti-GLI-1 (Cell Signaling, 2553), rabbit anti-AURKA (Cell Signaling, 4718), and mouse anti-β-actin (Sigma, A5441). Immunoreactivity bands were detected using a Pierce electrochemiluminescence western blotting substrate (Thermo Fisher Scientific).