Figure 6. Effect of increasing concentrations of TNF-α on the secretion of Complement C3 by ARPE-19 cells. Cells were treated with increasing concentrations of TNF-α ranging from 0 to 20 ng/ml for 24 h in serum-free media. Spent media were collected, and the levels of each protein were evaluated by western blot analysis. Panel A shows western blot against complement C3; both C3-α and C3-β subunits are detected. Panel B shows western blot against cytosolic IκB-α revealing activation of NF-κB signaling pathway. C: western blot against vinculin, a housekeeping protein whose level remained unchanged following TNF-α treatment. Panel D shows bar graphs of the relative expression of complement C3 to vinculin expression in function of different TNF-α concentration. Standard deviations were obtained from 3 replicate experiments with p-values <0.05.