(A-B) E2 induces Pak4 mRNA and protein levels. Serum-starved Ishikawa, RL95-2 and MCF-7 cells were treated with 10 nM E2, and cells were harvested at the indicated time. (A) The levels of Pak4 mRNA were determined by qRT-PCR, using β-actin as an internal control. Values represent mean ± s.d. (n = 3). ***P<0.001 compared with control. (B) The protein levels of Pak4 were assessed by Western blot. (C) Ishikawa and RL95-2 cells were serum-starved for 24 h, and then treated with 10 nM E2 for indicated times. The levels of p-Pak4ser474 were measured by Western blot, using β-actin as a loading control. All experiments were carried out in triplicates.