Fig 2: Purification of recombinant CCN proteins.(A) Analysis of purification of recombinant CCN2/CTGF from supernatants of stable transfected WB4 clone by SDS-PAGE and subsequent Coomassie stain. Bacterially expressed CCN2/CTGF (1 and 2 µg) served as an internal control. (B) Western blot analysis of individual fractions using a CCN2/CTGF-specific antibody. (C) Analysis of purification of CCN3/NOV from supernatants of COS-7 that were transiently infected with Ad-CMV-CCN3/NOV in Ponceau S stain. (D) The membrane from (C) was probed with a CCN3/NOV-specific antibody. (E) Different amounts (20 and 45 µg) of purified CCN3/NOV taken from fraction 2 (C, D) were analysed in Coomassie stain (left panel), Ponceau S stain (middle panel) and Western blot (right panel) with a CCN3/NOV-specific antibody.

Fig 3: Stability of purified recombinant CCN2/CTGF and CCN3/NOV.1 µg of purified CCN2/CTGF and CCN3/NOV that were kept for three months at indicated temperatures (4°C, -80°C) were separated on a NuPAGE Bis/Tris 4–12% gradient SDS gel under reducing and non-reducing conditions and stained with Coomassie. In this analysis 1 µg of rhCTGF (BioVendor) that was stored at -80°C served as an internal control.

Fig 4: MALDI-TOF analysis of purified recombinant CCN proteins.(A) Calibration spectrum of MALDI-TOF mass spectroscopy using a mixture of proteins that contains Trypsinogen, Protein A, and Albumin with average m/z 23982, 44613, and ~66500 as standards. (B) Mass spectrum of purified recombinant CCN2/CTGF. (C) Mass spectrum of purified recombinant CCN3/NOV.

Fig 5: 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-ß1, 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.