Effects of rhFNHN29 and rhFNHC36 on tyrosine phosphorylation of FAK and activation of AP-1. (A), Effect of PAO on the anti-adhesive activity of rhFNHN29 and rhFNHC36. MHCC97H cell suspension (2 × 105/100 μl) with or without Mn2+(0.1 mM), with or without PAO (20 μM) were pretreated with FN, rhFNHN29 and rhFNHC36 (200 μg/ml), respectively, and then seeded in a 96-well plate coated with or without FN (2.5 μg/ml). Adhesion of MHCC97H cells to the FN substrate was assayed under the indicated conditions as described in Figure 2. (B), Immunoblotting of p125FAK and EMSA of activated AP-1. MHCC97H cells were deprived of serum for 10 h, incubated in DMEM with or without PAO (20 μM) for 5 min, and further cultured with Mn2+(0.1 mM) in the presence or absence of FN, rhFNHN29 and rhFNHC36 (200 μg/ml) at 37°C for 20 min. The cytoplasmic protein and the nuclear protein were extracted respectively. The phosphotyrosyl proteins electroblotted to the PVDF membrane were visualized by anti-p-FAK (Tyr 397) after SDS-PAGE, and the activated AP-1 electroblotted to Nylon membrane was visualized by specific HRP-labeled probe after 6.5% PAGE for nuclear protein (F zone indicates non-binded free probe).