Ab225626

RIPA buffer was used to extract total protein. Proteins were quantified using a BCA protein determination Kit (KeyGEN Biotech, Nanjing, China). 30 μg protein was separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% non-fat milk for 1 h. The incubation of blots was conducted with antibodies at 4 °C overnight: Bax (ab32503, 1:1000, Abcam, Cambridge, MA, USA), Bcl-2 (ab117115, 1:1000, Abcam), ILF3 (ab225626, 1:1000, Abcam), AURKA (ab108353, 1:1000, Abcam), E2F1 (ab4070, 1:500, Abcam), E-cadherin (ab231303, 1:1000, Abcam), Vimentin (ab92547, 1:1000, Abcam), PI3K (#3811, 1:1000, CST, Danvers, MA, USA), phosphorylated PI3K (p-PI3K, #4228, 1:1,000, CST), AKT (#9272, 1:1,000, CST), phosphorylated AKT (p-AKT, #9271, 1:1000, CST) or GAPDH (ab8245, ab9485, 1:5000, Abcam) and then with HRP-conjugated second antibody (#7074, 1:1000, CST). Protein bands were detected with ECL Plus reagent (Pharmacia, Piscataway, USA), and visualized using a Gel Imaging System. Bands were then quantified using ImageJ software (National Institutes of Health). The expression of GAPDH was used for data normalization.

Cells were collected and lysed with 1 × NETN (0.5% NP-40, 20 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA) supplemented with proteinase inhibitor (cocktail, Roche). Cell lysates were collected by centrifugation and incubated with protein A/G beads (Sigma, U.S.) with the indicated antibodies for immunoprecipitation. Total proteins were obtained and separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE health, U.S.). The antibodies used in immunoprecipitation or western blotting were as follows: anti-Flag (F3165, Sigma, U.S.), anti-Myc (M4439, Sigma, U.S.), anti-CDK2 (ab32147, Abcam, U.K.), anti-cyclin E1 (ab33911, Abcam, U.K.), anti-cyclin B1 (sc-245, Santa cruz, U.S.), anti-NF90 (ab225626, Abcam, U.K.), anti-GAPDH (AP50812, Abgent, U.S.), anti-β-Actin (sc-47778, Santa cruz, U.S.) and anti-pNF-90 (S382; Youke, Shanghai, China). Roscovitine was purchased from Sigma and dissolved in dimethyl sulfoxide (DMSO) (Sigma, U.S.) and then included in the growth inhibition assay.
Antibodies to phosphorylated proteins were diluted by 5% BSA (Sigma, U.S.) in TBST according to the manufacturer’s instruction.