Figure 3

FLJ10540 expression and PI3K-FLJ10540 complex are restrained upon MLN8237 stimulation in HNC cells. (A and B) The mRNA and protein expression levels of FLJ10540 were examined by Q-RT-PCR and Western blotting in SAS cells in MLN8237 dose-dependent manner. The results were normalized against the expression level of GAPDH mRNA in each MLN8237-treated cell. Using the same panel, the total proteins were probed with antibodies against phosphorylated-Aurora-A, Aurora-A, FLJ10540 and β-actin. β-actin was used as a control. Data are representative of three independent experiments done in triplicate. (C) Luciferase assays were done to detect promoter activity of FLJ10540 in transfected FaDu cells in the presence or absence of MLN8237. (D) Unsynchronized HNC cells were fixed, and immunostained with antibodies against endogenous FLJ10540 or DAPI. Representative data are shown. (E) Co-immunoprecipitation of FLJ10540 and PI3K. Cells treated with or without MLN8237 and antibody of p110-α used for immunoprecipitation and representative western blot detection of p85-α and FLJ10540 were shown. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.