Ecl plus western blot system

Firstly, total proteins of Daudi and NAMALWA cells were extracted and quantified using BCA protein assay kit (Thermo Fisher Scientific, Cat. # A53227). Then proteins were separated via 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Next, samples were transferred to polyvinylidene difluoride (PVDF) membranes at 4 °C. After blocking, membranes were incubated first with primary antibodies (KIF15, Akt, p-Akt, CCND1, CDK6, PIK3CA and GAPDH) (Additional file 1: Table S1) and then with a secondary antibody (Goat Anti-Rabbit, 1:3000, Beyotime, Beijing, China, Cat. # A0208; Goat Anti-Mouse, 1:3000, A0216). Finally, immunoreactions were visualized using Amersham ECL + plusTM Western Blot system and the blots were imaged using a luminescent image analyzer.

After total proteins of U87 and U251 cells were extracted, quantified by BCA protein assay kit (Cat. No. A53227, Thermo Fisher Scientific, California, USA). CHX (0.2 mg/mL) refers to Cycloheximide blocking protein biosynthesis to study the half-life of FOXM1 and AURKA proteins over time (0–8 h). Same for the use of the proteasome inhibitor MG-132 (20 μM) to inquire about the protein degradation. Equivalent amount of protein was separated through 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) film at 4 °C. The protein was incubated with primary antibody and secondary antibody (antibody information was listed in Table S1) in turn at 4 °C for 3 h. The immune response was visualized with the Amersham ECL + plusTM Western Blot system, and the blots were imaged by luminescent image analyzer.