Figure 6. Lunasin alters the phosphorylation of RB.

Synchronized H661 cultures grown under anchorage-dependent growth conditions were treated with vehicle or 100 μM lunasin for 4 hours, and subsequently re-stimulated with medium containing 10 % serum in the presence of vehicle or 100 μM lunasin. Cells were harvested at the indicated times and protein extracts were isolated and subjected to SDS-PAGE and immunoblot analysis. A) Representative immunoblots of vehicle control and 100 μM lunasin exposed H661 cells probed with antibodies specific for total RB, and RB phosphorylated at S780, S807/811 and S608. β-actin was used for a loading control. B) Corresponding bar graphs of phosphorylated RB showing the delay in onset of RB phosphorylation. Immunoblots were analyzed using ImageJ software v1.45. Data represent the mean ± SD for three independent experiments. The asterisk (*) indicates a significant (p < 0.05) difference in protein phosphorylation levels when comparing control and lunasin treated cells at individual time points, while △ (control) and δ (lunasin) indicate a significant (p < 0.05) difference compared with the corresponding t = 0 measurement.