Figure 3.

LPA induces phosphorylation of Smad-2 during A431 colony dispersal. A431 cells were serum starved for 24 h and incubated with PBS or LPA (1, 2, or 4 μM) for 1 h. (a, b) Cells were lysed and 100 μg of total proteins were separated on 4–12% NuPAGE gels under reducing conditions. 3T3NIH cell lysate after TGF-β1 treatment was used as a positive control for phosphorylation of Smad2. After separation, the same gel was transferred to a PVDF membrane and blocked with 5% skim milk in 1X TBS and 0.1% Tween 20. After blocking, a pAb against phosphorylated Smad2 (p-Smad2; Ser465/467), a mAb against Smad2/3, or GADPH was added at the diluted ratio of 1 : 1000 and incubated at 4°C overnight. Anti-rabbit IgG or mouse IgG HRP-conjugated antibody was used as a secondary antibody at a diluted ratio of 1 : 1000. The bands were visualized with an ECL plus system. Protein expression levels were quantified from the western blots using ImageJ. Results showed that 4 μM LPA treatment for 12 h significantly enhanced pSmad2 protein expression compared to PBS treatment (N = 3; P =  .003).