Figure 4. Effects of bevacizumab and anti-CTGF antibody on MMP-2 and MMP-9 secretion by treated cultures. (A) Matrix metalloproteinase-2 (MMP-2) and (E) MMP-9 were detected and confirmed with western blot analysis of conditioned medium collected from human RPE cell culture without any treatment. To unravel the amount of MMP-2 and MMP-9 secretion, slot blot analysis was performed on media collected from the control and treated cultures. Briefly, equivalent amounts of concentrated collected media were transferred to nitrocellulose membranes with a slot blotter, blots were blocked, and then specified primary monoclonal antibodies were applied. The membrane was then incubated with secondary antibody, and bands were detected using the enhanced chemiluminescence (ECL) chemiluminescence system. Conditioned media collected from cultures incubated with standard medium, DMEM:F12 (1:1), was used as the control in all of the presented panels. In the cultures treated with 0.25, 0.5, and 0.8 mg/ml concentrations of bevacizumab, (B) MMP-2 and (F) MMP-9 increased whereas anticonnective tissue growth factor (anti-CTGF) application imposed a meaningful decrease in (C) MMP-2 or an increase in (G) MMP-9 secretion. The effect of the combined treatment of bevacizumab and anti-CTGF decreased (D) MMP-2 but did not affect (H) MMP-9 secretion *p<0.05. Results were quantified using the Quantity One software. Panel I; bars: 1, 2, and 3, Effect of cultures treated with 0.25, 0.5, and 0.8 mg/ml concentrations of bevacizumab on, MMP-2 secretion Panel I; bars: 4, 5, and 6, Effect of cultures treated with 0.25, 0.5, and 0.8 mg/ml concentrations of bevacizumab, on MMP-9 secretion Panel J (anti-CTGF treated culture compared to the control culture); bar: 1, anti-CTGF application imposed a meaningful decrease on MMP-2; Panel J; bar: 2, anti-CTGF application imposed a meaningful increase on MMP-9. Panel K; bar: 1, combined treatment of bevacizumab and anti-CTGF decreased MMP-2 Panel K; bar: 2, combined treatment of bevacizumab and anti-CTGF did not affect amount of MMP-9 in the culture media.