Figure 5.

Ephrin receptor A2 is increased in human prostatic metastatic tissue compared with primary CaP. (A) Invasion assays were performed using cell culture inserts (8 μℳ pore size) coated by a confluent layer of BMEC above a synthetic basement membrane (Matrigel). 1 × 10⁵ PC-3, PC3-GFP, DU 145 and LNCaP cells were added to the top of the inserts and allowed to invade for 18 h towards tissue culture plastic (TCP), BMS or AA (10 μℳ). (B) Western blot analysis showing EphA2 and EphrinA1 (EFNA1) content in PC-3, PC3-GFP, DU 145 and LNCaP cells. Loading was checked by immunoblotting the same samples with an anti-Actin antibody. Figure is representative of two separate experiments. (C) Lysates of DU 145 cells incubated with AA (10 μℳ) at different times were subjected to western blotting. Phosphorylation levels of Akt^s473, FAK^y576 and Src^y418 were compared with total levels of Akt, FAK and Src, respectively. Figures represent results of two separate experiments done in duplicate. (D) Lysates of primary cells from two separate patients with CaP and two patients with BPH were analysed by western blotting. Levels of EphA2 were compared with actin. Bands are representative of two separate experiments. (E) Invasion assays were performed using cell culture inserts (8 μm pore size) coated by a synthetic basement membrane (Matrigel). 1 × 10⁵ cells from CaP samples (n=2) and from BPH samples (n=2) were added to the top of the inserts and allowed to invade for 18 h towards AA (10 μℳ). Inserts were rinsed in PBS, fixed, stained in 2% crystal violet and counted. Results are the mean of two separate experiments.