Figure 8. Biological activity of purified recombinant CTGF, part II.

(A) EA•hy 926 cells were transfected with the (CAGA)₁₂-Luc reporter plasmid and incubated with indicated concentrations of recombinant human CCN2/CTGF. Cell lysates were prepared after 24 hr and luciferase activity determined. In this assay, the luciferase activity of normal control cells (0 ng/ml) was set to 1. The experiments were repeated three times. Shown are the means ±SD of one representative experiment performed in triplicate. (B) EA•hy 926 cells were transfected with the reporter plasmid and incubated with different concentrations of CCN3/NOV (500 or 1000 ng/ml) or PDGF (50 ng/ml) in serum-free medium. The promoter activity of the unstimulated controls was set as 1. The experiment was done three times in triplicate. The diagram depicts the means ± SD of one representative experiment. (C) After transfection with the Smad3 sensitive reporter, EA•hy 926 cells were incubated with recombinant CCN3/CTGF, a combination of CCN2/CTGF and CCN2/CTGF-specific antibody L-20, L-20 alone, or left untreated. 24 hr later, the relative luciferase activity was determined. The experiment was repeated three times. Shown are the results (means ±SD) of one representative experiment that was performed in triplicate. (D–G) EA•hy 926 cells were transiently transfected with the (CAGA)₁₂-Luc reporter plasmid and incubated with indicated concentrations/combinations of PDGF-BB, TGF-β1, CCN2/CTGF, CCN2/CTGF capturing antibody (L-20, sc-14939), and bacterial expressed CCN2/CTGF in serum-free medium. The promoter activity was measured 24 hr later.