Figure 4.

(a) Real-time PCR for 14-3-3 epsilon, RhoGDIα and DJ-1. Expression levels of 14-3-3 epsilon, RhoGDIα and DJ-1 was measured using quantitative real-time PCR with gene-specific primers given in Supplementary Table 3. Expression level of 18s was taken as an internal control for normalization of real-time PCR data. Experiments were performed in triplicate for each gene and the error bar represents s.d. *P<0.05 compared with vehicle-treated control cells and **P<0.001 compared with vehicle-treated control cells. (b) Western blot analysis of 14-3-3 epsilon, RhoGDIα, DJ-1, RhoA, Rac1 and Cdc42. NSCLC (A549) cells were treated with DAMTC (160 μg/ml) for 24 h. Total cell lysates were prepared and protein (40–60 μg) was subjected to SDS-PAGE, followed by immunoblot analysis using specific antibodies and secondary horseradish peroxidase-conjugated or alkaline phosphatase-conjugated antibodies. β-actin was used as internal control to ensure that equal amount of proteins were loaded in each lane. Blots are representative of three independent experiments with similar results. (c) Densitometric analysis of western blots was done as described in ‘Materials and Methods'. Data shown is representative of three independent experiments. The error bar represents s.d. *P<0.05 compared with vehicle-treated control cells